JPH0631288B2 - Polysaccharide hapten of human chorionic gonadotropin and its production method - Google Patents

Description

TECHNICAL FIELD The present invention relates to human chorionic gonadotropin (hereinafter referred to as “HC”).
G), a sugar chain-related polysaccharide hapten, a synthetic intermediate thereof, and a method for producing the same.

[Background of the Invention]

Human chorionic gonadotropin (HCG) is a hormone that stimulates the gonads. Since HCG is produced and secreted by the villous tissue of the placenta during pregnancy and appears in the urine of pregnant women in the early stages of pregnancy, it is used for determining pregnancy.

Further, HCG is a glycoprotein chemically having a subunit structure of α and β. Furthermore, a specific monoantennary sugar chain (A) has been found in the sugar chain isolated from the α-subunit of HCG of healthy humans except for sialic acid. On the other hand, H purified from the urine of patients with choriocarcinoma
It has been reported that CG contains an irregular biantennary sugar chain (B) [A. Kobata et al., Journal of Biological Chemistry (J. Bio
l. Chem. ), 258, 14126 (198)
3)].

Further, the modification of the sugar chain (A) to the sugar chain (B) is tentatively explained by the presence of N-acetylglucosaminyltransferase IV in the chorionic tumor tissue, but the details have been sufficiently elucidated. Absent. Therefore, the elucidation of the biological significance of such modifications that occur in glycoprotein sugar chains is currently under way.

From the viewpoint that it is indispensable to obtain an artificial carbohydrate antigen in order to elucidate the biological significance of the above-mentioned sugar chain modification, the present inventors have proposed a 7-sugar hapten (2) corresponding to the sugar chain (B). The present inventors have completed the present invention by carrying out the molecular design and the synthetic research.

[Object of the Invention]

The present invention relates to human chorionic gonadotropin (HCG)
Of the α-subunit asparagine-linked sugar chain, particularly a novel pentasaccharide and heptacylated hapten corresponding to the sugar chain (B) having a biantennary complex type structure, its synthetic intermediate, and its production method The purpose is to provide.

[Structure of Invention]

 That is, the novel compound of the present invention is represented by the following formula.

In the formula, R ¹ represents a hydrogen atom or a lower alkyl group, and R 1
² represents a hydrogen atom or a benzyl group, R ³ represents a hydrogen atom, an acetyl group or R ⁵ , R ⁴ represents a hydrogen atom, an acetyl group or R ⁵ , and R ⁵ represents R ⁶ represents a hydrogen atom, R ⁷ represents a hydrogen atom or an acetyl group, or R ⁶ and R ⁷ together represent a phthaloyl group, and R ⁸ represents a hydrogen atom or an acetyl group. Provided that when R ⁴ is a hydrogen atom or an acetyl group, R ³
Is not R ⁵ .

Specific examples of the present invention are shown below.

The compound (8) can be synthesized as follows. First, from allyl α-D-mannopyranoside (5), the method of Ogawa et al. [Carbohydrate Research, 1
14 , 225 (1983)] according to formula (6): (Wherein, X is a halogen atom, R ¹ is an acetyl group, and R ² is a benzyl group), and a compound (6) is synthesized. (In the formula, R ² represents a benzyl group and R ³ represents a lower alkyl group.) The compound (7) is reacted in the presence of a glycosylation catalyst to synthesize a compound (8). This glycosylation can be achieved by using AgOS in a solvent such as dichloroethane.
It is preferably carried out in the presence of a catalyst such as a silver salt such as O ₂ CF ₃ (also referred to as Ag triflate) or a mercury salt such as HgBr ₂ or a molecular sieve (hereinafter referred to as “MS”) 4A. This reaction is generally at -20 to 50 ° C,
It progresses sufficiently in 1 minute to 60 hours. This compound (8)
Is deacetylated with, for example, EtONa / EtOH to give compound (4).

This compound (4) was added to the known lactosaminyl halogenide (3) [M. M. Ponpipom et al.
Tetrahedron Letters, 1717 (1978), Ogawa et al., Carbohydrate Research, 97 , 81,
(1981) and the like] can be reacted in the presence of a glycosylation catalyst to give a 7-sugar compound (9) and a 5-sugar compound (17). This glycosylation reaction is also performed in a solvent such as dichloroethane using AgOSO ₂ CF.
₃ , silver salts such as HgBr ₂ , mercury salts such as MS4A, s
-In the presence of a catalyst such as collidine, -20 to 50 ° C, 1
Minutes to 60 hours is sufficient to proceed. The compound (9) and the compound are isolated by a conventional separation and purification means such as flash chromatography and gel permeation chromatography.

Compound (9) is treated with (i) LiOH and 31% H ₂ O ₂ aqueous solution in a solvent such as THF, then methanol solution such as NaOH, and (ii) NH ₂ NH in ethanol.
₂ · _{H 2} was treated with O: further (iii) in methanol,
By treatment with Ac ₂ O, compound (10), (1
Compound (13) is obtained through 1) and (12). Compound (13) was added to pd / in MeOH / AcOH, for example.
Catalytic reduction in the presence of C gives compound (14). This compound (14) was converted into (i) Ac ₂ O / pyridine, (ii)
CH ₂ N ₂ in THF / Et ₂ O: Further (iii) Na
By treating with OMe / MeOH, the target 7
A sugar hapten (2) is obtained.

Similarly, the compound (17) was converted into (i) THF in LiOH
And 31% H ₂ O ₂ aqueous solution, (ii) in ethanol,
Compound (21) is obtained via compounds (19) and (20) by treatment with NH ₂ NH ₂ · H ₂ O and then (iii) Ac ₂ O / pyridine. This compound (21) is deacetylated with, for example, MeONa / MeOH to give compound (22). Esterification of this compound (22) with diazomethane in MeOH / Et ₂ O gives compound (23) in high yield. This is further catalytically reduced in MeOH in the presence of pd / C,
The target pentasaccharide hapten (24) is obtained.

An example of the above steps is shown in the following scheme.

[Usefulness] The pentasaccharide and heptasaccharide haptens of the present invention can be chemically converted into antigens, and can be used for the diagnosis and examination of pregnancy and chorionic tumor. Can be used for. It is also a useful reagent for elucidating the biological significance of biantennary saccharides.

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1 8-Ethoxycarbonyloctyl 2,4-di-O-benzyl-3-O- (2,4-di-O-acetyl-3,6-
Di-O-benzyl-α-D-mannopyranosyl) -6-
O- (2,3,4,6-tetra-O-benzyl-α-D
Synthesis of -mannopyranosyl) -β-D-mannopyranoside (8) Compound (7) (1.06 g, 1 mmol), Ag triflate (385 mg, 1.5 mmol) and powdered molecular sieve 4A (2.0 g) were added to 1,2. Suspended in 10 m of dichloroethane. 6 m of a 1,2-dichloroethane solution of the compound (6) (0.64 g, 1.4 mmol) was added dropwise at 0 ° C-5 ° C. The mixture at 20 ° C. 1
After stirring for 6 hours, the mixture is diluted with 20 m of 1,2-dichloroethane and filtered through Celite. The filtrate is washed with saturated aqueous sodium hydrogen carbonate solution, then water, and dried over anhydrous magnesium sulfate. After concentration under reduced pressure, the residue is purified by silica gel column chromatography (3: 1, ethyl acetate-n-hexane) to obtain compound (8) (818 mg, 77.5%).

[Properties of Compound (8)] [α] _D −3.1 ° (c0.58, CHCl ₃ ), R _F
0.38 (2: 1, ethyl acetate-n-hexane) NMR: δ _H 5.524 (H-2c, dd, J1.5,
4Hz), 5.194 (H-1c, d, J1.5H
z), 5.146 (H-1b, d, J1.5 Hz),
4.294 (H-1a, s) , 2.251 (-CH 2 C
O ₂ Et, t, J7.3 Hz), 2.054 (CH ₃ C
_{O, s), 1.890 (CH} 3 CO, s), 1.7-
1.4 (6H, m), 1.35-1.15 (9H,
m). δ _C 101.6 (C-1a, ¹ J _CH 154 Hz), 9
9.7 (C-1c, ¹ J _CH 172 Hz), 98.6
(C-1b, ¹ J _CH 167 Hz) Elemental analysis calculated value (C ₈₉ H ₁₀₄ O ₂₀ .H ₂ O): C, 70.
71; H, 7.06 Experimental value: C, 70.81: H, 6.95 Example 2 8-ethoxycarbonyloctyl 2,4-di-O-benzyl-3-O- (3,6-di- O-benzyl-α-D-
Mannopyranosyl) -6-O- (2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl) -β-
Synthesis of D-mannopyranoside (4) Compound (8) (720 mg) is dissolved in 0.1 N sodium ethoxide ethanol solution 15 m and stirred for 16 hours at 20 ° C. The reaction solution is neutralized with Amberlyst A-15 and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (1: 1 ethyl acetate-n-hexane) to give compound (4) (672 mg, 99).
%).

[Properties of Compound (4)] [α] _D −0.8 ° (c1.1, CHCl ₃ ), R
_F 0.49 (1: 1, ethyl acetate-n-hexane) NMR: δ _H 5.226 (H-1c, d, J1.5H)
z), 5.141 (H-1b, d, J1.5Hz),
4.341 (H-1a, s), 4.116 4.116
(OCH ₂ CH ₃ , q, J7.1 Hz), 2.253
_{(CH 2 CO, t, J.7.6Hz} ), 1.65-1.
45 (4H, m), 1.35-1.15 (11H,
m). δ _C 101.6 (C-1a, ¹ J _CH 154H
z, C-1c, ¹ J _CH 169 Hz), 98.6 (C-
1b, ¹ J _CH 170 Hz), 60.0 (OCH ₂ CH
₃₎ Analysis Calculated _{(C 85 H 100 O 18)} : C, 72.42;
H, 7.15 Experimental value: C, 72.36: H, 7.14 Example 3 8-Ethoxycarbonyloctyl-2,4-di-O-benzyl-3-O- [3,6-di-O -Benzyl-2,4
-Di-O- {3,6-di-O-acetyl-2-deoxy-2-phthalimido-4-O- (2,3,4,6-tetra-O-acetyl-β-D-galactopyrano Sill) -β
-D-glucopyranosyl} -α-D-mannopyranosyl] -6-O- (2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl) -β-D-mannopyranoside (9) and 8- Ethoxycarbonyl octyl-
2,4-di-O-benzyl-3-O- [3,6-di-O
-Benzyl-4-O- {3,6-di-O-acetyl-2
-Deoxy-2-phthalimide-4-O- (2,3,
4,6-Tetra-O-acetyl-β-D-galactopyranosyl) -6-O- (2,3,4,6-tetra-O-benzyl-α-D-mannopyranosyl) -β-D- Synthesis of Mannopyranoside (17) Compound (4) (235 mg, 0.17 mmol), 2,
4,6-collidine (97.0 μ, 0.73 mmol)
And Ag triflate (188 mg, 0.73 mmo
1) is dissolved in 6 m of 1,2-dichloroethane.

Compound (3) (524 mg, while stirring at -15 ° C)
0.66 mmol) of 1,2-dichloroethane solution 6 m
Is dripped. After stirring the mixture at -15 ° C for 1 hour,
Dilute with 20 m of 1,2-dichloroethane and filter through Celite. The filtrate is washed with water, a saturated sodium hydrogen carbonate aqueous solution and then with water, and then dried over anhydrous magnesium sulfate. After concentration under reduced pressure, the residue is purified by silica gel column chromatography (3: 2, toluene-ethyl acetate) to obtain compound (17) (115 mg, 32.6%).

[Properties of Compound (17)] [α] _D + 14.4 ° (c1.05, CHCl ₃ ), R
_F 0.44 (1: 1, toluene-ethyl acetate) NMR: δ _H 5.648 (H-3d, dd, J7.5).
8.9 Hz), 5.647 (H-1d, d, J8.5H
z), 5.315 (H-4e, d, J = 3.0 Hz),
5.124 (H-1b, or H-1c, d, J1.5H
z), 5.116 (H-1c, or H-1b, d, J
1.5 Hz), 5.076 (H-2e, dd, J8.
0, 10.4 Hz), 4.918 (H-3e, q, J
3.4, 10.4 Hz), 4.468 (H-1e, d,
J7.7 Hz), 4.218 (H-1a, s), 2.1.
31 (CH ₃ CO), 2.027 (CH ₃ CO), 1.
973 (CH ₃ CO), 1.970 (CH ₃ CO),
1.959 (CH ₃ CO), 1.876 (CH ₃ C
O), 1.7-1.4 (4H, m), 1.3-1.1
(11H, m) Elemental analysis Calculated value (C ₁₁₇ H ₁₃₅ O ₃₅ N): C, 66.4
3; H, 6.43; N, 0.66 Experimental value: C, 66.23: H, 6.39; N, 0.66 The column was further eluted with the same solvent system and the fraction containing compound (9) was continued. (367 mg) was obtained. This gives a single spot of R _F 0.34 on TLC for 1: 1 toluene-ethyl acetate, but R _F 0.29 and R _F 0.16 for 1: 2: 1 n-hexane-ethyl acetate-dichloroform.
The two spots are given in a ratio of about 1: 1. This fraction was further used as a solvent in benzene and 200 cm x 2 cm.
The compound (9) (174 mg) and 1,3,6-tri-O-acetyl-2-deoxy-2-phthalimido-4-O were separated and purified by gel filtration chromatography using the bio-beads of Bio-Beads. −
(2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl) -α-D-mannopyranose (17
0 mg, R _F 0.29) was obtained. The latter was mixed in the brominated body (3) from the beginning.

[Properties of Compound (9)] [α] _D + 2.5 ° (C1.1, CHCl ₃ ), R
_F 0.16 (1: 2: 1 n-hexane-ethyl acetate-
Chloroform) NMR: δ _H 5.577 (H-1f, d, J8.5H)
z), 5.561 (H-3d, dd, J8.5, 10.0H
z), 5.400 (H-3e, dd, J8.5, 9.0H)
z), 5.314 (H-4f, d, J4.0Hz), 5.290 (H-4g, d, J4.0Hz), 5.092 (H-2f, dd, J8.0, 10.0H).
z), 5.057 (H-1b, and H-1c, d, J1.5H
z), 5.035 (H-2g, dd, J8.0, 10.4H
z), 2.136 (CH ₃ CO), 2.119 (CH ₃ C
O), 2.060 (CH ₃ CO), 2.058 (CH ₃
CO), 2.021 (CH ₃ CO), 1.973 (CH
₃ CO), 1.950 (CH ₃ CO × 3), 1.844 (CH ₃ C)
O) 1.827 (CH ₃ CO), 1.685 (CH ₃ CO) Elemental analysis calculated value (C ₁₄₉ H ₁₇₀ O ₅₂ N ₂ ): C, 63.4
4; H, 6.07; N, 0.99 Experimental value: C, 62.90; H, 6.03; N, 0.99 Example 4 8-ethoxycarbonyloctyl 3-O- [2-O- Acetyl-3,6-di-O-benzyl-4-O- {3,6
-Di-O-acetyl-2-deoxy-2-phthalimido-4-O- (2,3,4,6-tetra-O-acetyl-
β-D-galactopyranosyl) -β-D-glucopyranosyl} -α-D-mannopyranosyl] -2,4-di-O
-Benzyl-6-O- (2,3,4,6-tetra-O-
Synthesis of benzyl-α-D-mannopyranosyl) -β-D-mannopyranozide (18) Compound (17) (3.0 mg) was dissolved in 0.5 m of a 1: 1 mixture of pyridine and acetic anhydride, and the mixture was stirred at 25 ° C for 18 hours. Stir. Concentrated under reduced pressure and purified by silica gel chromatography (1: 1 toluene-ethyl acetate) to give compound (1
8) is obtained.

[Properties of Compound (18)] R _F 0.64 (1: 1 toluene-ethyl acetate) NMR: δ _H 5.430 (H-2c, dd, J1.5,
_{3Hz), 2.121 (CH 3 CO} ), 2.027 (CH 3 C
O), 2.000 (CH ₃ CO), 1.951 (CH ₃
CO), 1.944 (CH ₃ CO), 1.928 (CH
_{3 CO), 1.859 (CH 3} CO) Example 5 8-methoxycarbonyloctyl 3-O-[4-O-
{2-acetamido-2-deoxy-4-O-galactopyranosyl) -β-D-mannopyranosyl} -α-D-mannopyranosyl] -6-O- (α-D-mannopyranosyl) -β-D-mannopyranoside Synthesis of (24) Compound (17) (31.0 mg, 0.015 mmol)
Is dissolved in 1 m of tetrahydrofuran and stirred at 0 ° C. Add 31% hydrogen peroxide solution (0.24m),
After stirring at 0 ° C. for 5 minutes, a 1.25N aqueous lithium hydroxide solution (89μ, 0.11 mmol) is added. 0 ℃ ~
Stir at 20 ° C. for an additional hour and add the reaction mixture to Amberlyst A.
Neutralize with -15. It was diluted filtered through Celite with ethyl acetate 10 m, after washing the filtrate, dried over anhydrous magnesium sulfate, concentrated under reduced pressure and the compound (19) (14.5 mg) to obtain the _[R F 0.36 (6: 1 chloroform - methanol) ]. Compound (19) (14.0 mg, 0.008 m
mol) and hydrazine hydrate (20 μ) are dissolved in 4 m of ethanol, and the mixture is heated under reflux for 18 hours. Concentration under reduced pressure gives compound (20). ] R _F 0.23 (6: 1 chloroform-methanol)]. Compound (20) is dissolved in pyridine-acetic anhydride 2: 1 (3 m) and stirred at 20 ° C. for 20 hours. After concentration under reduced pressure, the residue is purified by silica gel column chromatography (1: 1 toluene-ethyl acetate) to obtain compound (21) (11.0 mg, 70%).

[Compound (21) the nature of] R _F 0.26 (1: 1 toluene - ethyl _{acetate) NMR: δ H 2.14 (CH} 3 CO), 2.04 (C
H ₃ CO), 2.02 (CH ₃ CO), 1.97 (CH
₃ CO × 2), 1.96 (CH ₃ CO), 1.69 (C
H ₃ CONH) Compound (21) (17 mg) was dissolved in 0.1 N sodium methoxide methanol solution 4 m, and the solution was dissolved at 20 ° C. for 18
The mixture is stirred for an hour, neutralized with Amberlyst A-15, and concentrated under reduced pressure to give compound (22) (13 mg, 90%).

[Properties of Compound (22)] R _F 0.21 (6: 1 chloroform-methanol) NMR: δ _H (CD ₃ OD) 1.82 (CH ₃ CON)
H) Compound (22) (12.5 mg) was dissolved in 4 m of methanol, a diazomethane ether solution was added, and the mixture was added at 20 ° C. to 2.
Process for 5 hours. After decomposing excess diazomethane with a methanol solution of acetic acid and concentrating under reduced pressure, the residue is purified by silica gel column chromatography (6: 1 chloroform-methanol) to obtain compound (23) (12 mg 95%).

[Compound (23) the nature of] R _F 0.21 (6: 1 chloroform - _{methanol) NMR: δ H (CD 3} OD) 3.63 (OMe), 1.
82 _(CH 3 CONH) Compound (23) (12.0mg) and 10% palladium - carbon (10 mg) was suspended in methanol, stirred for 4 hours under 20 ° C. a hydrogen atmosphere. After filtration through Celite, the filtrate is concentrated under reduced pressure, and the residue is purified by Sephadex G-25 (water) to obtain the compound (24) (2 mg).

[Compound (24) the nature of] _{R F 0.44 (2: 1:} 1 n- butanol - ethanol - _{water) NMR: δ H (D 2} O.50 ℃) 5.111 (H-1
c, bs), 4.902 (H-1b, bs), 4.66
1 (H-1a, s), 4.585 (H-1d, d, J
8.2Hz), 4.463 (H-1e, d, J7.6H)
z), 3.683 (OMe, s), 2.050 (CH ₃
CONH) Example 6 8-oxycarbonyloctyl 3-O- [2,4-di-O- {2-acetamido-2-deoxy-4-O- (β
-D-galactopyranosyl) -β-D-glucopyranosyl} -α-D-mannopyranosyl] -6-O- (α-D
-Mannopyranosyl) -β-D-mannopyranoside (1
Synthesis of 4) 1.2 m of 31% hydrogen peroxide aqueous solution is added to 5 m of tetrahydrofuran solution of compound (9) (130 mg, 0.05 mmol), and the mixture is stirred at 0 ° C for 5 minutes. 1.25N lithium hydroxide aqueous solution (0.525m, 0.66mmo
1) is added and the mixture is stirred at 0 ° C.-25 ° C. for 3 hours.
Amberlyst A-15 is added to neutralize the reaction solution, insoluble matter is filtered off, and then treated with excess dimethyl sulfide. After concentration under reduced pressure, purification by column chromatography using Sephadex LH-20 (methanol) is performed to obtain a mixture of compound (10) and its monoacetyl derivative (87 mg, 82%). R _F 0.57 and 0.6
5 (2: 1: 1 n-butanol-ethanol-water) NMR: δ _C (CD ₃ OD) 14.9 (OCH ₂ C
H ₃ ); δ _H (CD ₃ OD) 1.95 (CH ₃ CO) Crude product (10) (60 mg, obtained as above)
0.026 mmol) in 6 m of methanol solution 0.1
0.4 m of N sodium hydroxide aqueous solution is added, and the mixture is stirred at 20 ° C. for 16 hours. After diluting the reaction solution with 6m of a 1: 1 mixture of tetrahydrofuran-water, Amberlyst A
Add -15 to neutralize. The insoluble material is filtered off and concentrated under reduced pressure to give compound (11) (55 mg) as a crude product. _{R F 0.55 (2: 1:} 1n- butanol - ethanol - water) Compound (11) (55 mg) and hydrazine hydrate (0.
5m) is dissolved in 12m of ethanol and the mixture is heated to reflux. 48 hours later, hydrazine hydrate (0.5 m)
Is added and the mixture is heated under reflux for further 60 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified with Sephadex LH-20 (methanol) to give compound (11) (44 mg, 90%).
To get R _F 0.43 (2: 1: 1 n-butanol-
Ethanol-water) Compound (13) (44 mg) was dissolved in a mixed solution of methanol (5 m) and acetic anhydride (1 m), and the mixture was mixed at 20 ° C for 1 hour.
Stir for 6 hours. After concentration under reduced pressure, the residue was purified with Sephadex LH-20 (methanol) to give compound (13) (4
4 mg) is obtained. R _F 0.58 (2: 1: 1 n-butanol-ethanol-water), R _F 0.22 (40:20:
3 Chloroform-methanol-water) Compound (13) (43 mg) and 10% palladium-carbon were suspended in a mixed solution of methanol (4 m) and acetic acid (0.4 m), and stirred at 50 ° C for 3 hours under a hydrogen atmosphere. To do. The reaction solution is filtered through Celite, and the filtrate is concentrated under reduced pressure.
The residue is purified by Sephadex G-25 (water) to obtain the compound (14) (13 mg, 43%) as an amorphous powder.

[Properties of Compound (14)] [α] _D +4.3 ° _{(c0.5, H 2 O) R} F 0.17 (2: 1: 1 n- butanol - ethanol - water) NMR: [delta] _H (D ₂ O, 20 ° C) 5.110 (H-1
c, s), 4.891 (H-1b), 4.648 (H-
1a, s), 4.551 (H-1d or H-1e, d, J7.6H)
z), 4.532 (H-1e or H-1d, d, J7.8H)
z), 4.452 (H-1f and H-1g, d, J7.8H)
z), 4.205 (H-2c, bs), 4.086 (H-2)
a, bs), 2.060 (CH ₃ CONH), 2.034 (CH ₃ C)
ONH) Elemental analysis calculated value (C ₅₅ H ₉₄ O ₃₈ N ₂ · H ₂ O): C, 4
6.87; H, 6.86 Experimental value: C, 46.56; H, 6.78 Example 7 8-methoxycarbonyloctyl 3-O- [2,4-
Di-O- {2-acetamido-2-deoxy-4-O-
(Β-D-galactopyranosyl) -β-D-glucopyranosyl} -α-D-mannopyranosyl] -6-O- (α
Synthesis of -D-mannopyranosyl) -β-D-mannopyranoside (2) Compound (14) (6.5 mg, 4.6 μmo) was dissolved in pyridine (2 m) and acetic anhydride (1 m), and 25
Stir at 16 ° C for 16 hours. After concentration under reduced pressure, the residue was purified by Sephadex LH-20 (methanol) and the compound (1
5) is obtained (10.2 mg, 98%).

R _F 0.23: - Add (24 1 chloroform-methanol) Compound (15) with excess diazomethane ether solution in tetrahydrofuran solution 2m in (10.2 mg) at 0 ° C.. While the reaction is only spot with the same R _F is observed and after the 16 hours standing at 0 ℃ compounds on even thin layer chromatography (15), compound (16) was obtained by usual post-treatment .

The compound (16) is dissolved in 3 m of a methanol solution of 0.1N sodium methoxide and stirred at 20 ° C. for 3 hours.
Neutralization with Amberlyst A-15, the insoluble matter was filtered off, and the mixture was concentrated under reduced pressure to obtain a mixture (5.3 mg) of compound (2) and compound (14). R _F 0.23 [compound (2)]
And 0.17 [compound (14)] (n-butanol-ethanol-water). The mixture was purified by preparative thin layer chromatography (2: 1: 1 n-butanol-ethanol-water) to give compound (2) (1.8 mg) and compound (1).
4) (2.2 mg) is obtained.

[Properties of Compound (2)] NMR: δ _H (D ₂ O, 20 ° C) 5.110 (H-1)
c, s), 4.893 (H-1b, s), 4.649 (H-1a,
s), 4.551 (H-1d or H-1e, d, J7.3H)
z), 4.532 (H-1e or H-1d, d, J8.3H)
z), 4.454 (H-1f and H-1g, d, J7.8H)
z), 4.204 (H-2c, bs), 4.098 (H-2)
a, bs), 3.673 (OMe), 2.376 (CH ₂ CO, t,
J7.3 Hz), 2.059 (CH ₃ CONH), 2.033 (CH ₃ C
ONH), 1.584 (4H, m), 1.4-1.2 (11H,
m)

Claims (6)

[Claims] 1. A compound represented by the following formula: In the formula, R ¹ represents a hydrogen atom or a lower alkyl group, and R 1
² represents a hydrogen atom or a benzyl group, R ³ represents a hydrogen atom, an acetyl group or R ⁵ , R ⁴ represents a hydrogen atom, an acetyl group or R ⁵ , and R ⁵ represents R ⁶ represents a hydrogen atom, R ⁷ represents a hydrogen atom or an acetyl group, or R ⁶ and R ⁷ together represent a phthaloyl group, and R ⁸ represents a hydrogen atom or an acetyl group. Provided that when R ⁴ is a hydrogen atom or an acetyl group, R ³
Is not R ⁵ . 2. The compound according to claim (1), wherein R ¹ is an ethyl group, and R ³ and R ⁴ are hydrogen atoms. ^3. The compound according to claim (1), wherein R ³ and R ⁴ are R ⁵ . 4. R ³ is a hydrogen atom or an acetyl group,
The compound according to claim (1), wherein R is R ⁵ . 5. Formula (6): (Wherein, X represents a halogen atom, R ¹ represents an acetyl group, R ² represents a benzyl group), and a compound represented by the formula (7): (Wherein R ² represents a benzyl group and R ³ represents a lower alkyl group), the compound (7) is reacted in the presence of a glycosylation catalyst, and optionally acetylated and deprotected. formula: (Wherein R ¹ represents a hydrogen atom or an acetyl group, R ² represents a hydrogen atom or a benzyl group, and R ³ represents a hydrogen atom or a lower alkyl group). 6. A formula (3) (In the formula, X is a halogen atom, R is an acetyl group, Phth
Represents a phthaloyl group) and a compound (3) represented by the formula (4) (Wherein R ¹ represents a lower alkyl group and R ² represents a benzyl group), the compound (4) is reacted in the presence of a glycosylation catalyst, and optionally acetylated and deprotected. Expression: (In the formula, R represents a hydrogen atom or an acetyl group, R ¹ represents a hydrogen atom or a lower alkyl group, R ² represents a hydrogen atom or a benzyl group, and R ³ represents a hydrogen atom, an acetyl group or Wherein R ⁴ represents a hydrogen atom, R ⁵ represents a hydrogen atom or an acetyl group, or R ⁴ and R ⁵ jointly represent a phthaloyl group).