JPH0631290B2 - HCG sugar chain-related pentasaccharide paptene and method for producing the same - Google Patents

Description

TECHNICAL FIELD The present invention relates to a sugar chain-related pentasaccharide hapten of human chorionic gonadotropin (hereinafter referred to as HCG) 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.

In addition, 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 .alpha.-subunit of HCG of a healthy person 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) (1983).

Furthermore, the modification of the sugar chain (A) to the sugar chain (B) is tentatively explained by the presence of N-acetylglucosaminyl transferase IV in chorionic tumor tissue, but details are sufficient. Has not been elucidated. 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 biophysical significance of the above-mentioned sugar chain modification, the present inventors have considered that a pentasaccharide peptene (V) corresponding to the sugar chain (A) is used. The present invention has been completed by conducting the molecular design of (1) and conducting synthetic research thereof.

[Object of the Invention]

It is an object of the present invention to provide a novel pentasaccharide paptene corresponding to an asparagine-linked sugar chain of α-subunit of human chorionic gonadotropin, particularly a sugar chain having a monoantenna type complex structure. .

[Structure of Invention]

That is, the novel pentasaccharide peptene of the present invention is a compound represented by the following general formula (V).

(In the formula, R ¹ represents a benzyl group or a hydrogen atom, and R ² represents an acetyl group or a hydrogen atom.) Further, the novel compound of the present invention has the following general formula (I),
It is represented by (II), (III) and (IV).

(In the formula, R represents a hydrogen atom or a —CH ₂ CH═CH ₂ group, and Bn
Represents a benzyl group, and Et represents an ethyl group. ) (In the formula, R represents a benzyl group and Et represents an ethyl group.) (In the formula, R represents a benzyl group and Et represents an ethyl group.) (In the formula, R ¹ represents a —CH ₂ CH═CH ₂ group, R ² represents a benzyl group, R ³ represents a phthaloyl group or an acetyl group, R ⁴ represents a phthaloyl group or a hydrogen atom, and R 2 ⁵ represents an acetyl group, or R ¹ represents a hydrogen atom or a —C (═NH) CCl ₃ group, R ² represents a benzyl group, R ³ and R ⁵ represent an acetyl group, and R ⁴ represents Hereinafter, the present invention will be described in detail.

(a) Synthesis of Compound of General Formula (IV) As shown in Scheme 1, the compound (12) represented by the general formula (IV) is a known compound, allyltri-O-benzyl-α-D-mannopyranozide (10). ) [T. Ogawa et al., Carbo Hydro. response. (Carbohydr. Res.), 9
3 (1983) C3-C9] is a known compound N-phthaloyllactosyl bromide (11) [M. M. Ponpipom et al., Tetrahedron Lett., (197
8) It can be produced by the method of 1717-1720]]. This glycosylation is preferably carried out in a solvent such as Cl (CH ₂ ) ₂ Cl in the presence of AgOSO ₂ CF ₃ and molecular sieve (hereinafter abbreviated as MS) 4A. Also, the glycosylation reaction is preferably carried out at a temperature of −20 to 50 ° C. for 1 minute to 60 hours, for example, by stirring.

Further, the compound (13) is synthesized by deacetylating the compound (12) and then dephthaloylating.

Deacetylation of the compound (12) can be carried out by treating with NaOCH ₃ in a solvent such as methanol. After the deacetylation, it can be neutralized with Amberlyst 15 if necessary.

Also, the dephthaloylation subsequent to the deacetylation is preferably carried out by treating the deacetylated product with BuNH ₂ in a solvent such as methanol and then with acetic anhydride / pyridine.

Further, the compound (14) is synthesized by deallylating the compound (13). This deallylation is preferably carried out by treating compound (13) with Pdcl ₂ and sodium acetate in acetic acid. Deallylation is
It is preferably carried out by stirring the reaction system at a temperature of −10 to 100 ° C. for 5 minutes to 48 hours.

Compound (15) is synthesized by converting compound (14) into chlorimidate. This chlorimidate conversion is carried out by converting compound (14) into NaH and C in a solvent such as methylene chloride.
It is preferably carried out by treating with l ₃ CCN. Furthermore, the chlorimidate conversion reaction is -60 to 50 ° C.
Is preferably carried out under ice cooling for 5 minutes to 18 hours while stirring the reaction system.

(b) Synthesis of Compound of General Formula (I) The compound (18) represented by the general formula (I) can be prepared by using the scheme 2
Compound (17) [R.
U. Lamieux et al. Am.Chem. Soc., 9
7 (1975) 4076-4083] can be produced by glycosylation with a known compound, compound (16). This glycosylation is preferably carried out, for example, in a solvent such as dichloroethane in the presence of silver silicate as well as powdered MS4A. The glycosylation reaction is -50 to 100 ° C.
Can be performed by stirring the reaction system at the temperature of 5 minutes to 70 hours.

The above compound (18) [β-D-mannopyranosyl C
-9 ester] and its isomer, compound (19) [α-D-mannopyranose C-9 ester].
Is generated. The compounds (18) and (19) are separated by a means such as column chromatography.

Further, the compound (20) is obtained by deallylating the compound (18). This deallylation is carried out by the compound (2
It is preferable to carry out step 0) by treating with PdCl ₂ and sodium acetate in acetic acid. The temperature of the deallylation reaction is −
It is preferably carried out by stirring the reaction system at 10 to 100 ° C. for 5 minutes to 96 hours.

(c) Synthesis of compounds of the general formulas (II) and (III) The compound (23) represented by the general formula (II) and the general formula (II)
The compound (22) represented by I) is synthesized by glycosylating the compound (20) with a known compound, tetra-O-benzylmannopyranosyl chloride (21). This glycosylation is preferably carried out in the presence of Bu ₄ NCl in a solvent such as dichloroethane and the reaction is carried out in an argon atmosphere at a temperature of 0 to 100 ° C.
It is preferable to carry out heating under reflux for 240 hours.

(d) Synthesis of Compound of General Formula (V) The compound (24) represented by the general formula (V) is a compound (2)
3) is synthesized by glycosylating compound (15). This glycosylation is preferably carried out in the presence of BF ₃ .Et ₂ O in a solvent such as dichloroethane. Furthermore, the glycosylation reaction is preferably carried out in an atmosphere of argon or the like at a temperature of -20 to 100 ° C for 5 minutes to 48 hours while stirring the reaction system.

Further, the compound (25) is synthesized by reducing the compound (24) with hydrogen. This hydrogen reduction is carried out in the presence of Pd-C in a hydrogen gas atmosphere in a solvent such as acetic acid at 5 to 100 ° C.
It is preferable to carry out at the temperature of 30 minutes to 100 hours.

Then, the compound (26) is synthesized by deacetylating the compound (25). This deacetylation is preferably carried out by treatment with NaOEt in a solvent such as ethanol. The deacetylation reaction is preferably carried out at a temperature of −5 to 50 ° C. for 30 minutes to 16 hours while stirring the reaction system.

(12): R ¹ = -CH ₂ CH = CH ₂ , R ² = Bn, R ³ , R ⁴ = phthaloyl group, R ⁵ = Ac (13): R ¹ = -CH ₂ CH = CH ₂ , R ² ＝ Bn, R ³ , R ⁵ ＝ Ac, R ⁴ ＝
^{H (14): R 1 =} R 4 = H, R 2 = Bn, R 3 = R 5 = Ac (15): R 1 = C (= NH) CCl 3, R 2 = Bn, R 3 = R 5 = Ac, R ⁴
= H The compounds (12) and (1
3), (14), (15), (18), (19), (2
0), (22), (23), (24), (25), (2
All of 6) are novel compounds.

〔Usefulness〕

The pentasaccharide peptene (V) of the present invention can be chemically converted into an antigen and can be used for the preparation of an antibody that can be used for examination of pregnancy.

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

Example 1 (compound (10) + (11) → (12)) 3.95 g (8.07 mmol) of compound (10) was added to dichloroethane 2
Dissolve in 0 ml and add AgoTrif. 3.45 g (13.44 mmol) and syn-
1.77 ml of collidine (13.44 mmol) was added, and 5.15 g (6.72 mm) of N-phthaloyllactosyl bromide (compound (11)) was added while cooling and stirring at -20 ° C under an argon atmosphere.
ol) in dichloroethane. After stirring at the same temperature for 20 minutes, the reaction solution is diluted with methylene chloride and filtered through Celite. The filtrate is a 10% HCl solution, saturated sodium bicarbonate solution,
The extract is washed with saturated saline and dried over MgSO ₄ . After concentration under reduced pressure, the residue is silica gel (Wako gel C-300,5
The compound (12) (3.41 g, 83%) was obtained by purification with 00 g, toluene: ethyl acetate = 3: 1). (Compound (1
2.27 g of 0) was recovered. ) [Α] ²⁵ _D ▼ −27 ° C = 1.28 CHCl ₃ Elemental analysis Calculated value (C ₆₂ H ₆₉ NO ₂₃・ 1 / 2C ₆ H ₅ CH ₃ ) C, 63.33, H, 5.92, N, 1.13 Measured value C, 63.32, H, 5.95, N, 1.16. Rf = 038 Toluene: Ethyl acetate = 3: 1 Example 2 (Compound (12) → (13)) Compound (12) 3.4 g (2.84 mmol) was added to methanol 25 ml.
The resulting mixture is dissolved in water, 2.0 ml of N-NaOCH ₃ methanol solution is added, and the mixture is stirred at room temperature for 17 hours. The reaction solution is neutralized with Amberlyst 15, filtered, and the latter solution is concentrated under reduced pressure. . The residue is dissolved in 150 ml of methanol, 30 ml of n-butylamine is added, and the mixture is heated under reflux for 60 hours. After cooling, the reaction solution was concentrated under reduced pressure,
75 ml of pyridine and 75 ml of acetic anhydride are added to the residue, and the mixture is stirred at room temperature for 18 hours. The reaction solution was concentrated under reduced pressure, toluene was added to the residue for azeotropic distillation to remove the solvent, and the residue was purified with a silica gel column (Wakogel C-300 500 g toluene: ethyl acetate = 2: 3) to give compound (13). obtain.

Rf = 0.32 Toluene: Ethyl acetate = 1: 2 ▲ [α] ²⁵ _D ▼ + 2.8 C = 0.53 CHCl ₃ Elemental analysis Calculated value C, 60.69, H, 6.28, N, 1.26 Measured value C, 60.56, H, 6.30 , N, 1.38 Example 3 (Compound (13) → (14)) Compound (13) 300 mg (0.27 mmol) was added to 95% acetic acid 3 ml.
Dissolve in, add 53 mg (0.30 mmol) of palladium chloride and 49 mg (0.60 mmol) of sodium acetate, and stir at 70 ° C. for 1 hour. After cooling, the reaction solution is filtered through Celite and concentrated under reduced pressure. The residue was applied to a silica gel column (Wako Gel C-300).
Purification with 50 g chloroform: acetone = 3: 1) gives compound (14) (182 mg, 63%).

Rf = 0.54 Chloroform / acetone 1: 1 ▲ [α] ²⁷ _D ▼ -10.62 C = 1.44, CHCl ₃ Elemental analysis Calculated value (C ₅₃ H ₆₅ NO ₂₂ ) C, 59.59, H, 6.13, N, 1.31 Measured value C , 59.60, H, 6.19, N, 1.22 Example 4 (Compound (14) → (15)) 500 mg (0.468 mmol) of compound (14) was dissolved in 18 ml of methylene chloride, and 1.8 ml of trichloroacetonitrile and NaH were stirred under ice cooling. Add 22.4 mg (50% oily 0.933 mmol) and stir for 3 hours. The reaction solution was filtered through Celite and concentrated under reduced pressure, and the residue was filtered through a silica gel column (Wako Gel C-300).
Purified with toluene: ethyl acetate = 1: 3, the compound (1
5) (540 mg, 95%).

Rf = 0.54 Toluene: Ethyl acetate = 1: 3 ▲ [α] ²⁰ _D ▼ + 9.08 C = 1.09, CHCl ₃ Example 5 (Compound (16) → (18) + (19)) 5.0 g of activated MS4A Compound (17) 1.01 g (5.0
mmol), silver silicate 5.0 g, dichloroethane 2
0 ml was added, a 10 ml solution of the compound (16) in dichloroethane was added dropwise with stirring under ice cooling, and the mixture was gradually returned to room temperature for 2 hours 3 hours.
Stir for 0 minutes. The reaction solution is diluted with 250 ml of ethylene chloride, filtered through Celite, and the filtrate is washed with saturated sodium bicarbonate water, washed with water and dried over magnesium sulfate. After concentration under reduced pressure, the residue was purified by a silica gel column (Wakogel C-3
00 300 g Toluene-ethyl acetate 10: 1), and the compound (18) (β-D-mannopyranosyl C-9 ester) (2.19 g, 67.9%) and its isomer, compound (19) (α). -D-mannopyranosyl C
-9 ester) (0.53 g, 16.9%) was obtained.

Compound (18) ▲ [α] ²⁷ _D ▼ -44.3 C = 1.01 CHCl ₃ Rf = 0.41 Toluene: Ethyl acetate = 10: 1 Elemental analysis Calculated value (as C ₃₇ H ₅₂ O ₈ ) C, 71.12, H, 8.39 measurement Value C, 70.87, H, 8.34 Compound (19) ▲ [α] ²⁷ _D ▼ + 13.1 ° C = 2.06 Rf = 0.52 Toluene: Ethyl acetate = 10: 1 Elemental analysis Calculated value (as C ₃₇ H ₅₇ O ₈ ) C, 71.12, H, 8.39 Measured value C, 70.89, H, 8.31 Example 6 (compound (18) → (20)) Compound (18) 5.25 g (8.4 mmol) was added to 95% acetic acid 30 ml.
Dissolved in, 3.28 g (18.4 mmol) of PdCl ₂ , sodium acetate
Add 3.32 g (40.4 mmol) and stir at 80 ° C. for 1 hour.
After cooling, the reaction solution is filtered through Celite and concentrated under reduced pressure. The residue is dissolved in acetic ester, washed with sodium hydrogen carbonate solution and saturated brine, dried over MgSO ₄ , and concentrated under reduced pressure. The residue was purified by a silica gel column (Wakogel C-300 300 g hexane / EtoAc 2: 1) to give compound (20) (3.47).
g, 73.3%).

Rf = 0.56 Hexane: EtoAc = 1: 1) ▲ [α] ²⁷ _D ▼ +4.63 C = 0.97 CHCl ₃ Elemental analysis Calculated value (as C ₃₁ H ₄₄ O ₈ ) C, 68.36, H, 8.14 Measured value C, 68.00, H, 8.11 Example 7 (compound (20) + (21) → (22) +
(23)) 4.52 g of compound (20) was added to 18.0 g of activated MS4A.
(7.52 mmol), nBu ₄ NCl 4.55 g (19.9 mmol) dissolved in 90 ml of dichloroethane and added, and the compound (21),
A solution prepared by dissolving 6.0 g (10.7 mmol) in 30 ml of dichloroethane was added dropwise, and the mixture was heated under reflux for 3 hours under an argon atmosphere. After adding 30 ml of dichloroethane and further heating under reflux for 48 hours, the reaction solution is filtered, the filtrate is washed with saturated sodium bicarbonate water and saturated saline, dried over MgSO ₄ , and concentrated under reduced pressure. The residue was passed through a silica gel column (Wako Gel C
-300 500 g, hexane: ethyl acetate = 2: 1)
The compound (22) (0.78 g, 10.2%) and the compound (23) (3.93 g, 77.2%) are obtained after purification and separation by. Furthermore, 1.5 g (Rf = 0.24) of unreacted compound (20) was recovered.

Compound (22) ▲ [α] ²⁸ _D ▼ + 9.75 ° C = 1.63 CHCl ₃ Rf = 0.45 Hexane: ethyl acetate = 2: 1 Elemental analysis Calculated value C, 74.79, H, 7.10 Measured value C, 74.63, H, 7.26 Compound (23) ▲ [α] ²⁸ _D ▼ -4.5 ° C = 1.14 CHCl ₃ Rf = 0.38 Hexane: ethyl acetate = 2: 1 Elemental analysis Calculated value C, 72.60, H, 7.36 Measured value C, 73.10, H, 7.36 Example 8 (Compound (15) + (23) → (24)) Activated MSAW-300 350 mg, Compound (15)
131 mg (0.108 mmol) Compound (23) 131 mg (0.122
mmol) dissolved in 3 ml of dichloroethane and added with stirring under ice cooling
132 μ (0.108 mmol) of BF ₃ · Et ₂ O is added, and the mixture is stirred in an argon atmosphere for 1 hour. After the reaction solution is filtered through Celite, the filtrate is diluted with ethyl acetate and washed with sodium bicarbonate water. The extract was dried over MgSO ₄ , dried under reduced pressure, and the residue was purified by silica gel column (Wakogel C-300 40 g toluene: ethyl acetate = 1: 1) to give compound (24).
(102 mg, 45%) is obtained.

Rf = 0.26 Toluene: Ethyl acetate = 1: 1 ▲ [α] ²⁷ _D ▼ -8.7 ° C = 0.36 CHCl ₃ Elemental analysis Calculated value C ₁₁₈ H ₁₄₁ O ₃₄ N ・ 4H ₂ O C, 64.73, H, 6.49, N , 0.64 Measured value C, 64.62, H, 6.42, N, 0.85 Example 9 (Compound (24) → (25) → (26)) 124 mg (0.058 mmol) of the compound (24) was dissolved in 6 ml of acetic acid to obtain 10%. Add 70 mg of Pd-C, and in a hydrogen gas atmosphere,
Catalytic reduction is performed at 60 ° C. for 6 hours. The reaction solution is filtered through Celite and concentrated under reduced pressure to obtain a debenzylated product (compound (25)). 76mg (100%) Rf = 0.64 (n · BuOH: EtOH: H 2 O = 4: 2: 2).

▲ [α] ²⁸ _D ▼ -2.6 C = 0.96, CHCl ₃ 70 mg (0.053 mmol) of debenzylated product obtained here was dissolved in 5 ml of absolute ethanol, and N-NaOCH ₃ methanol solution was added.
Add 0.1 ml and stir at room temperature for 18 hours. The reaction solution is neutralized with Amberlyst-15 and concentrated under reduced pressure. 48 mg (85
%). This substance is purified by column chromatography on Sephadex G-25 to obtain compound (26).

[Α] _D + 4.9 ° C = 0.425 H ₂ O Rf = 0.44 n ・ BuOH: EtOH: H ₂ O = 4: 2: 2 Elemental analysis Calculated value (as C ₄₃ H ₇₅ NO ₂₈ ) C, 44.44, H , 6.50, N, 1.20 Measured value C, 44.54, H, 6.52, N, 1.56

Claims (2)

[Claims] 1. A compound represented by the general formula (V). (In the formula, R ¹ represents a benzyl group or a hydrogen atom, R ² represents an acetyl group or a hydrogen atom, Ac represents an acetyl group,
Et represents an ethyl group. ) 2. A compound (15) represented by general formula (IV) [In the general formula (IV), R ¹ represents a —C (═NH) CCl ₃ group, R ² represents a benzyl group, R ³ and R ⁵ represent an acetyl group, and R ⁴ represents a hydrogen atom. ] And a compound represented by the general formula (II) (23) [In the general formula (II), R represents a benzyl group. ] The compound represented by the general formula (V) is reacted, and if necessary, deprotected. (In the formula, R ¹ represents a benzyl group or a hydrogen atom, R ² represents an acetyl group or a hydrogen atom, Ac represents an acetyl group,
Et represents an ethyl group. )