JPH0689041B2 - Method for producing oligogalacturonic acid - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing oligogalacturonic acid.

[Conventional technology]

It is well known that plants protect themselves by inducing relatively low-molecular-weight phytoalexin when invading pathogens and parasites, and research is progressing in legumes and solanaceous plants. I'm out. However, the plant-physiological part is currently being actively pursued, and many problems still remain for elucidating the whole picture at the molecular level. Accumulation of phytoalexin was not found in healthy plants, and it was not produced until the invasion of the fungus, so it is clear that some kind of chemical stimulus was involved. Chemical substances that cause such irritation are called elicitors, and it has been confirmed that some organic substances and some inorganic substances perform this function. In particular, those derived from the former include enzymes produced by pathogenic bacteria, cell wall-derived substances of pathogenic bacteria, and host plant cell wall-derived polysaccharides.
Albershim et al. Found a substance having elicitor activity in a hot water extract of soybean cell wall and a partial acid hydrolyzate of the cell wall, and its structure was α1 →
It was clarified that it is a galacturonic acid oligomer that is linearly linked by 4 bonds [M G. Hahn, A. G. Dalville, P. Albersheim, Plant Physiol.
(Plant Physiology) 68, 1161-1169 (1981)]. Partial acid hydrolysis of citrus pectin also yields elicitors [EA Nosnagel, M Muknile, P. Albersheim, Adel, Plant.
Physiol. (Plant Physiology) Vol. 71, 916-926 (1983); Reference 1], has elicitor activity even when partially hydrolyzed with an enzyme (endopolygalacturonic acid lyase) produced by pathogenic bacteria such as soybean cell wall and pectin. It was revealed that a substance was obtained [K. Earl Davis, A.
Gee Dalville, Pee Albersheim, Adel, Z. Naturforsch (Naphthilhosk, Volume 41c, 39-
48 (1986)].

West et al. Also reported the formation of galacturonic acid oligomers and the elicitor activity by enzymes of pathogenic bacteria using castor beans [RJ.
Brace, C.A. West, Plant Physiol. (Plant Physiology), 69, 1181-1188 (1982)].

Fractions showing activity are represented by structural formula I (n = 7 to 12) (R =
H).

Among the partial enzymatic hydrolysis products, Albersheim et al. Report that those of structural formula II also have strong activity.

It is extremely important to find out what kind of molecular structure (unit) is effective as an active elicitor for phytoalexin production and how it functions in the plant, Precise synthesis of galacturonic acid oligomers is indispensable because it is very difficult to obtain a large amount of purer samples than in the natural world. Furthermore, it becomes possible to investigate in detail the mechanism of phytoalexin production using the galacturonic acid oligomer obtained in this way, and by knowing this, a new way of controlling biological production using the plant's self-defense ability can be opened. Is expected.

[Problems to be Solved by the Invention]

An object of the present invention is to provide a method for producing an oligogalacturonic acid represented by the formula (2), which has an activity as an elicitor for inducing phytoalexin production of an antibacterial substance existing in the cell wall of a plant. Is.

The present inventors have already reported that in the general formula (I), R is β-n.
A method for synthesizing a 10-sugar oligogalacturonic acid (1) having a -propyl group and n = 9 has been established (Japanese Patent Application No. 62-227496).
Compound), which was pointed out by Albersheim et al. To have the highest activity as a phytoalexin elicitor (general (I), wherein R is hydrogen and n is a dodecasaccharide). The present invention was completed by conducting research for the purpose of synthesizing oligogalacturonic acid).

Unlike the compound (1), this compound (2) has a hemiacetal structure similar to that obtained from the reducing terminal part of the oligomer, and as described above, Albers
It is a compound pointed out by him et al. to have the highest activity as a phytoalexin elicitor (Reference 1).

[Means for Solving the Problems]

The synthesis of compound (1), the reducing end as a precursor was performed using as a protecting group allyl group _{(-CH 2 CH = CH 2)} , the protecting group At a oligo galacturonic acid Since it was revealed in the reaction using the model compound that it was difficult to remove selectively, the t-butyldiphenylsilyl group (Si (C ₆ H ₅ ) ₂ But) was used as an alternative protecting group. The synthetic route was pioneered. In addition, in order to improve the efficiency of oligomer synthesis, we have newly developed a method of condensing oligomer blocks that are larger than those used in the synthesis of compound (1). That is, tetrasaccharide oligomers such as (3), (4), and (5) are prepared separately, and these are first prepared in (4) and (5),
Next, the target compound (2) was synthesized by condensing with (3).

That is, according to the present invention, a compound (4) represented by the formula (4) and a compound (5) represented by the formula (5) are condensed to obtain an octasaccharide compound (16), which is deisopropylidated. After preparing the compound (17), the 6-position is selectively acetylated to form an octasaccharide sugar acceptor (18), and this is combined with the tetrasaccharide sugar donor (3) represented by the formula (3). A formula characterized by condensing to obtain a 12-sugar compound (19), deacetylating this to form a compound (20), then oxidizing it to lead to a carboxylic acid (21), and removing a protecting group. This is a method for producing a 12-sugar oligogalacturonic acid (2) represented by (2).

A compound (3) used as a starting material in the method of the present invention,
Both (4) and (5) are novel compounds, and their synthesis method will be described first.

In the present specification, Bn represents a benzyl group, Ac represents an acetyl group, Ph represents a phenyl group, and t-Bu represents a t-butyl group.

Synthesis of Compound (3) Known Compound (6) ((Y.Nakahara, T.Ogawa Tetrakedon
Lett. (1987) 28 2731-2734); Reference 2) is deisopropylidated with 80% acetic acid to give compound (7), which is then selectively acetylated to obtain compound (8). This compound (8) and known compound (9) (reference 2) under the conditions of Mukaiyama et al. (T. Mukaiyama, Y. Murai, S. Shoda, Chem. Lett., (1981)
p.431-432) Condensate to give compound (10). Compound (1
0) to Schmidt's conditions (W.Kinzy, RRSchmidt, Juetus L.
iebigs Ann, Chem., (1985) 1537-1545) under desilylation and then DAST in THF (W. Rosenbrook, Jr., DARiley, PALa
rtey, Tetraledron-Lett., (1985) 26 3-4G.H.Posner, S.
R. Harnes, Terahedron Lett., (1985) 26 5-8) to give a fluoride mixture (3) with α: β = 7: 18.

Synthesis of Compound (4) The known compound (12) (Reference 2) is condensed with the compound (8) under the conditions described above to obtain the compound (13). Compound (13) is desilylated as before to give compound (14) and further treated with DAST to lead to a fluoride mixture (4) of α: β = 7: 18.

Synthesis of Compound (5) The above-mentioned Compound (13) is deisopropylidated to give Compound (5), which is selectively acetylated to obtain Compound (5).

Condensation of blocks (3), (4), and (5) (synthesis of dodecasaccharide) Condensation of the above-mentioned compounds (4) and (5) gives an octasaccharide compound (16), which is deisopropylidene Compound (17) and then selectively acetylated to give an octasaccharide sugar acceptor (18), which undergoes a condensation reaction with a tetrasaccharide sugar donor (3) to produce a dodecasaccharide compound (19). To get

Conversion of Compound (19) to Oligogalacturonic Acid (2) Compound (19) is deacetylated using sodium methoxide in methanol as a catalyst to obtain Compound (20). to this
Swern oxidation (K.Omura, D.Swern, Tetrahedron (1978) 34 1
651-1662) followed by sodium chlorite oxidation (GAKr
aus, B.Roth J.Org, Chem., (1980) 45 4825-4830) leads to a carboxylic acid (21). This is desilylated by the above-mentioned method to give compound (22), and then debenzylated by hydrogenolysis to obtain target compound (2).

When the compound (21) was converted into the corresponding methyl ester, further purified, and heated to LiI in pyridine to return it to the compound (21) again, the crude product (2) obtained in the final step was obtained. Purity is improved.

Separate Synthesis of Octasaccharide Compound (17) Compound (17) can be synthesized by the following method. That is, the hexasaccharide compound (23) is obtained by condensing the compounds (12) and (5), which is deisopropylidated to the compound (24), and then selectively acetylated to give the compound (25).
And then condensed again with the compound (12) to obtain the compound (16), and the crude product (16) is deisopropylidated to obtain the compound (17). This is in perfect agreement with the one synthesized by the previous method.

As a reagent for the reaction for deisopropylidating compounds (6), (13), (16) and (23), hydrous acetic acid, trifluoroacetic acid and the like can be used, and hydrous acetic acid is preferred.

Further, as the catalyst, p-toluenesulfonic acid, sulfuric acid, hydrochloric acid or the like can be used.

Further, as the solvent, tetrahydrofuran, water, methanol, ethanol or the like can be used, and tetrahydrofuran is preferable.

The reaction can also be carried out at room temperature to about 80 ° C., preferably at room temperature, for about 0.5 hours to about 24 hours, preferably 17 hours.
Sufficiently progress by carrying out the stirring reaction for a time.

The reaction product thus obtained is purified by a conventional method such as column chromatography.

As a reagent for the reaction of partially acetylating the compounds (7), (15), (17) and (24), acetyl chloride, acetic anhydride, acetylimidazole or the like can be used, and preferably acetyl chloride.

In addition, pyridine or triethylamine is used as the base.

Further, as the solvent, pyridine, dichloromethane, ether or the like can be used, and anhydrous pyridine is preferable.

Further, this reaction can be carried out at about −10 ° C. to room temperature, preferably ice cooling, for about 2 hours to about 2 days, preferably 1.
Sufficiently progress by carrying out a stirring reaction for 5 hours.

The reaction product thus obtained is purified by a conventional method such as column chromatography.

Compounds (8) and (9), (8) and (12), (4) and (5), (13) and (18), (5) and (12), (25) and (12), etc. As a catalyst for the glycosylation reaction, stannous chloride, molecular sieves 4A powder, silver perchlorate, silver triflate or the like can be used.

As the solvent, ether, toluene, dichloromethane, dichloroethane, or the like can be used, and anhydrous ether is preferable.

Further, this reaction is carried out at about -15 ° C to about 60 ° C, preferably 0 ° C.
The reaction can be carried out at 0 ° C., and the reaction proceeds for about 1 hour to about 24 hours, preferably 2.5 hours with stirring.

The reaction product thus obtained is purified by a conventional method such as column chromatography.

As a reagent for the desilylation reaction of the compounds (10), (13) and (21), tetra-n-butylammonium fluoride,
Acetic acid or pyridine-hydrogen fluoride and the like can be used.

Further, as the solvent, tetrahydrofuran, acetonitrile, dichloromethane, ether or pyridine can be used.

Further, this reaction can be carried out at about −20 ° C. to about 60 ° C., preferably room temperature, and is sufficiently progressed by carrying out the stirring reaction for about 10 hours to about 100 hours, preferably 52 hours.

The reaction product thus obtained is purified by a conventional method such as column chromatography.

As a reagent for the reaction for fluorinating the compounds (11) and (14), diethylaminosulfatrifluoride (DAST) or pyridine-hydrogen fluoride can be used, and diethylaminosulfatrifluoride is preferable.

Further, as the solvent, tetrahydrofuran, toluene,
Carbon tetrachloride, ether, dichloromethane, chloroform or the like can be used, and anhydrous tetrahydrofuran is preferable.

Further, this reaction can be carried out at about −30 ° C. to about 60 ° C., preferably room temperature, and proceeds sufficiently by carrying out a stirring reaction for about 0.1 hour to about 10 hours, preferably 0.5 hour.

The reaction product thus obtained is purified by a conventional method such as column chromatography.

As a reagent for the reaction for deacetylating compound (19),
Sodium methoxide, sodium ethoxide, sodium hydroxide or potassium hydroxide can be used, preferably 0.1N sodium methoxide.

As the solvent, methanol, ethanol, tetrahydrofuran or the like can be used, and preferably methanol-
It is anhydrous tetrahydrofuran.

Further, this reaction can be carried out at about −5 ° C. to about 60 ° C., preferably room temperature, and is sufficiently progressed by carrying out a stirring reaction for about 1 hour to about 2 days, preferably 24 hours.

The reaction product thus obtained is purified by a conventional method such as column chromatography.

The reaction conditions for oxidizing compound (20) to obtain compound (21) are as follows.

(First Step) First, as a reaction reagent, oxalyl chloride-dimethylsulfoxide, triethylamine, diisopropylethylamine, trichloroacetic anhydride, trifluoroacetic anhydride, acetic anhydride, dicyclohexylcarbodiimide-dimethylsulfoxide, or the like can be used.

As the solvent, dichloromethane, dichloroethane, tetrahydrofuran or dimethylsulfoxide can be used, preferably dichloromethane or dimethylsulfoxide.

Furthermore, this reaction can be carried out at about -78 ° C to room temperature, preferably at a cooling temperature, and it proceeds sufficiently by carrying out a stirring reaction for about 0.1 hour to about 24 hours, preferably 0.2 hour.

(Second Stage) First, as the catalyst, sodium chlorite, monosodium phosphate, sodium hypochlorite, chromic acid-sulfuric acid or the like can be used.

As the solvent, t-butyl alcohol, 2-methyl-2-butene, acetonitrile, water, tetrahydrofuran or acetone can be used, and t-butyl alcohol and 2-methyl-2-butene are preferably used in combination.

Further, this reaction can be carried out at about −10 ° C. to about 60 ° C., preferably room temperature, and is sufficiently progressed by carrying out a stirring reaction for about 3 hours to about 2 days, preferably 12 hours.

The reaction product thus obtained is purified by a conventional method such as column chromatography.

Diazomethane or the like can be used as a reagent for the reaction of compound (21) for methyl esterification.

In addition, ethanol or the like can be used as the solvent.

The reaction product thus obtained is purified by a conventional method such as column chromatography.

As a catalyst for the debenzylation reaction for reducing the compound (22) with hydrogen to obtain the compound (2), palladium carbon, Raney nickel or the like can be used, preferably 10%
Palladium on carbon.

Further, as the solvent, methanol, ethanol, water-
Methanol, acetic acid-water or the like can be used, and water-containing methanol is preferable.

Also, this reaction is carried out at about 15 ° C to about 80 ° C, preferably at room temperature
Can be carried out for about 1 to about 10 days, preferably
Sufficiently progress by carrying out a stirring reaction for 4 days.

The reaction product thus obtained is purified by a conventional method such as column chromatography.

Hereinafter, the present invention will be described in more detail with reference to Examples and Examples, and the contents thereof are as follows.

Reference Example 1 t-butyldiphenylsilyl O- (2,3-di-O-benzyl-α-D-galactopyranosyl)-(1 → 4)-
6-O-acetyl-2,3-di-O-benzyl-β-D-
Galactopyranoside (7) Dissolve known compound (6) (1.37g, 1.34mmol) in tetrahydrofuran (5ml) and add 80% hydrous acetic acid (30ml) to it.
Was added and the mixture was stirred at room temperature for 2 days. After concentration under reduced pressure, the residue was purified by column chromatography using silica gel (120 g). Elution with toluene-ethyl acetate (3: 2) gave compound (7) (1.20 g, 1.22 mmol, 91.2%).

[Properties of Compound (7)] [α ▲] ²⁷ _D ▼ + 54.0 (c, 1.4, CHCl ₃ ) ¹ H-NMR (400 MHz, CDCl ₃ ) δ 1.14 [3 H, s, -C (C
H _{3) 3],} 1.88 (3H, s, COC H 3), 3.27 (1H, t, J = 6.
34Hz, H-5a), 3.31 (1H, dd, J = 10.01,2.68, H-3a), 3.5
2 (1H, m, H-6b), 3.62 (1H, dd, J = 1.47,5.13Hz, H-6
b), 3.70 (1H, dd, J = 10.01,7.32, H-2a), 3.78 (1H, d,
J = 2.44Hz, H-4a), 3.88 (1H, dd, J = 9.77,3.41Hz, H-2
b), 3.97 (1H, dd, J = 9.77,3.18Hz, H-3b), 4.07 (1H,
m, H-5b), 4.09 (1H, m, H-4b), 4.15 (1H, dd, J = 11.23,
6.59Hz, H-6a), 4.24 (1H, dd, J = 11.23,6.35Hz, H-6
a), 4.60 (1H, d, J = 7.32Hz, H-1a), 4.61 (1H, d, J = 1)
1.72Hz, -C H ₂ Ph), 4.68 (1H, d, J = 11.72Hz, -C H ₂ P
h), 4.71 (1H, d, J = 12.0Hz, -C H ₂ Ph), 4.76 (1H, d, J
= 11.72Hz, -CH ₂ Ph), 4.80 (1H, d, J = 12.0Hz, -C H ₂ P
h), 4.83 (1H, d, J = 11.72Hz, -C H ₂ Ph), 4.84 (1H, d, J
= 11.0Hz, -C H ₂ Ph), 4.91 (1H, d, J = 3.41Hz, H-1b),
4.96 (1H, d, J = 11.0Hz, -C H ₂ Ph), 7.24-7.42 (26H, m,
Aromatic), 7.72 (4h, m, Aromatic). ¹³ C-NMR (22.5 MHz, CDCl ₃ ) δ 19.29, 20.70, 27.1
4, 62.69, 63.12, 69.46, 70.11, 72.55, 73.09, 73.7
9, 75.09, 76.12, 76.33, 77.48, 80.73, 98.28 (C-1
a), 100.24 (C-1b), 127.27, 127.43, 127.54, 127.8
1, 127.92, 128.14, 128.30, 128.41, 128.52, 129.5
5, 133.66, 135.78, 135.99, 137.95, 138.11, 138.6
0, 170.23 (C = 0).

Elemental analysis Analytical value C, 70.47H; H, 6.74 Calculated value (C ₅₈ H ₆₆ O ₁₂ Si) C, 70.85; H, 6.77 Reference Example 2 t-butyldiphenylsilyl O- (6-O-acetyl-2,3 -Di-O-benzyl-α-D-galactopyranosyl)-(1 → 4) -6-O-acetyl-2,3-di-O-
Benzyl-β-D-galactopyranoside (8) Diol (7) (1.38 g, 1.4 mmol) was added to anhydrous pyridine (15).
Acetyl chloride (125 ml)
μ, 1.76 mmol, 1.25 eq) was added. 3 at 0 ℃ to 10 ℃
After stirring for an hour, water (1 ml) was added to stop the reaction, and the mixture was concentrated under reduced pressure. The residue was extracted with ethyl acetate-ether (1: 1), washed with water and saturated brine, and dried over anhydrous sodium sulfate. The crude product obtained by distilling off the solvent was purified by column chromatography using silica gel (150 g). Elution with toluene-ethyl acetate (3: 1) was followed by a small amount (0.1 g, 6.7%) of triacetate [t-butyldiphenylsilyl O- (4,6-di-O-acetyl-2,3-di-O]. -Benzyl-α-D-galactopyranosyl)-(1
→ 4) -6-O-acetyl-2,3-di-O-benzyl-
β-D-galactopyranoside] {Rf0.56 (7: 3 = toluene: EtOAc), ¹ H-NMR (90 MHz, CDCl ₃ ) δ1.13 [9 H, s,-
C (C H ₃ ) ₃ ], 1.88 (3H, s, COC H ₃ ), 1.92 (3H, s,
COC H ₃ ), 2.08 (3H, s, COC H ₃ ), 5.56 (3H, m, H-4
b)}, and then compound (8) (1.25 g, 1.2 mmol, 86.9
%) Was obtained.

[Compound (8) the nature] Rf 0.35 (7: 3 = toluene: EtOAc), [alpha ▲] _{^{23 D ▼ + 55.1 (c,}} 0.7, CHCl 3) 1 H-NMR (500MHz, CDCl 3) δ 1.12 [9H, s, -C (C
H _{3) 3],} 1.88 (3H, s, COC H 3), 1.90 (3H, s, COC H
₃ ), 3.26 (1H, t, J = 6.57Hz, H-5a), 3.30 (1H, dd, J =
9.76,2.75Hz, H-3a), 3.71 (1H, dd, J = 9.76,7.33Hz, H-2
a), 3.81 (1H, d, J = 2.44, H-4a), 3.89 (1H, dd, J = 9.7
6,3.36Hz, H-2b), 4.01 (1H, dd, J = 9.76,3.05Hz, H-3
b), 4.04 (1H, dd, J = 10.98,6.11Hz, H-6b), 4.05 (1H,
br.s, H-4b), 4.13 (1H, dd, J = 1.30,7.02Hz, H-6a),
4.19 (1H, dd, J = 11.29,7.02Hz, H-6a or H-6b), 4.23
(1H, dd, J = 11.29,6.11Hz, H-6a or H-6b), 4.35 (1H,
t, J = 6.41Hz, H-5b), 4.60 (1H, d, J = 7.33Hz, H-1a),
4.65 (1H, d, J = 12.82Hz, -C H ₂ Ph), 4.70 (1H, d, J = 11.
90Hz, -C H ₂ Ph), 4.72 (1H, d, J = 12.82Hz, C H ₂ Ph), 4.
78 (1H, d, J = 1.29Hz, -C H ₂ Ph), 4.80 (1H, d, J = 1.90
Hz, -C H ₂ Ph), 4.83 (1H, d, J = 1.90Hz, -C H ₂ Ph), 4.8
6 (1H d, J = 10.98Hz, -C H ₂ Ph), 4.94 (1H, d, J = 3.36H
z, H-1b), 4.96 (1H, d, J = 10.98Hz, -C H ₂ Ph), 7.22-7.
42 (26H, m, aromatic), 7.69-7.42 (4H, m, aromatic). ¹³ C-NMR (22.5 MHz, CDCl ₃ ) δ 19.29, 20.70, 20.8
1, 27.09, 62.69, 63.01, 67.56, 68.11, 72.33, 72.5
5, 73.85, 75.04, 75.47, 76.18, 77.37, 80.24, 80.7
8, 98.07 (C-1a), 99.75 (C-1b), 127.22, 127.43, 1
27.54, 127.92, 128.14, 128.30, 129.49, 135.78, 13
5.99, 138.16, 138.27, 138.65, 170.45 (C = 0), 17
0.23 (C = 0).

Elemental analysis Analytical value C, 70.27; H6.69 Calculated value (C ₆₀ H ₆₈ O ₁₃ Si) C, 70.29; H, 6.69% Reference Example 3 t-butyldiphenylsilyl O- (6-O-acetyl-2,3 , 4-Tri-O-benzyl-α-D-galactopyranosyl)-(1 → 4) -O- (6-O-acetyl-2,3
-Di-O-benzyl-α-D-galactopyranosyl)-
(1 → 4) -O- (6-O-acetyl-2,3-di-O-
Benzyl-α-D-galactopyranosyl)-(1 → 4)
-6-O-Acetyl-2,3-di-O-benzyl-β-D
-Galactopyranoside (10) Stannous chloride (155mg, 0.82mmol), silver perchlorate (185m
g, 0.89 mmol) and well dried Monocular Sieves 4A powder (1.0 g) under an argon stream under cooling in an ice-methanol bath and stirring with compound (8) (200 mg,
A solution of 0.20 mmol) and (9) (225 mg, 0.26 mmol, 1.3 eq) in anhydrous ether (17 ml) was applied. Bath temperature -10 ° to 10 ° C
After stirring the reaction solution for 3 hours, the reaction was stopped by adding pyridine (2 ml), and the insoluble material was filtered over Celite. This was further washed well with ethyl acetate-ether (1: 1), the organic layers were combined, washed with water and saturated saline, and then dried over anhydrous sodium sulfate. The crude product obtained by distilling off the solvent was purified by column chromatography on silica gel (70 g) and developed with toluene-ethyl acetate (4: 1) to develop compound (10) (337 mg, 0.18 mmol, 91.7%). ) Got.

[Properties of Compound (10)] [α ▲] 25 D ▼ + 47.6 (c, 0.8, CHCl 3 ) 1 H-NMR (500 MHz, CDCl 3 ) δ 1.12 [9 H, s, -C (C
H 3) 3], 1.78 (3H, s, COC H 3), 1.89 (3H, s, COC H
3), 1.90 (3H, s , COC H 3), 1.93 (3H, s, COCH 3), 3.
22 (1H, br.t, H-5a), 3.25 (1H, dd, J = 9.77,2.75Hz, H-3
a), 3.64 (1H, dd, J = 9.77,7.32Hz, H-2a), 3.70 (1H, d
d, J = 10.37Hz, 4.58), 7.20-7.46 (51H, m, aromatic), 7.68-7.72 (4H, m, aromatic). 13 C-NMR (22.5 MHz, CDCl 3 ) δ 19.23 [ -C (CH 3 )
3], 20.64 (CO C H 3) , 20.81 (CO C H 3), 27.04 [-
C (C H 3) 3], 97.96 (C-1a, 1 J CH 161.1Hz), 99.37 (
1 J CH 167.2Hz), 99.69 ( 1 J CH 168.5Hz) (C-1b, c, d), 1
69.75 (C = 0 x2), 169.91 (C = 0), 170.23 (C =
0).

Elemental analysis Analytical value C, 70.78; H, 6.54 Calculated value (C ₁₁₁ H ₁₂₂ O ₂₅ Si) C, 70.76; H, 6.53% Reference Example 4 O- (6-O-acetyl-2,3,4-tri- O-benzyl-α-D-galactopyranosyl)-(1 → 4) -O-
(6-O-acetyl-2,3-di-O-benzyl-α-D
-Galactopyranosyl)-(1 → 4) -O- (6-O-
Acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl)-(1 → 4) -6-O-acetyl-2,3-
Di-O-benzyl-D-galactopyranose (11) The silyl compound (10) (101 mg, 0.54 mmol) was dissolved in anhydrous tetrahydrofuran (1.8 ml), and glacial acetic acid (28 µ, 0.5
mmol), then 1M tetra-n-butylammonium fluoride-tetrahydrofuran solution (0.25 ml, 0.25 mol)
Was added and the mixture was stirred at room temperature for 6 days. The mixture was diluted with an ether-ethyl acetate (1: 1) mixed solvent (50 ml), washed with water and saturated saline, and dried over anhydrous sodium sulfate.
The crude product obtained by distilling off the solvent was purified by column chromatography using silica gel (10 g), and toluene-
Develop with ethyl acetate (7: 3) and compound (11) (79 mg, 0.0
5 mmol, 89.5%) was obtained as an anomeric mixture.

[Properties of Compound (11)] ¹ H-NMR (500 MHz, CDCl ₃ ) δ 1.776 (1.780) (3 H, s,
_{COC H 3), 1.883 (1.901} ) (3H, s, COCH 3), 1.937 (1.
940) (3H, s, COC H ₃ ), (2.031) 2.033 (3H, s, COC H 3
₃ ), 5.26 (H-1a, α-anomer). ^{13 C-NMR (22.5MHz, CDCl} 3) δ 20.59 (C H 3), 20.7
5 ( C H ₃ ), 91.35 (C-1a, α-anomer), 97.58 (C-1
a, β-anomer), 99.31 & 99.58 (C-1b, c, d), 169.75
(C = 0), 169.85 (C = 0), 170.13 (C = 0), 17
0.40 (C = 0).

Elemental analysis analysis C, 69.26; H, 6.34 Calculated _{(C 95 H 124 O 25)} C, 69.22; H, 6.37% Reference Example 5 O- (6-O- acetyl-2,3,4-tri -O -Benzyl-α-D-galactopyranosyl)-(1 → 4) -O-
(6-O-acetyl-2,3-di-O-benzyl-α-D
-Galactopyranosyl)-(1 → 4) -O- (6-O-
Acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl)-(1 → 4) -6-O-acetyl-2,3-
Di-O-benzyl-D-galactopyranosyl fluoride (3) Hemiacetal (11) (130 mg, 79 μmol) was dissolved in anhydrous tetrahydrofuran (1 ml), cooled on an ice-methanol bath, and stirred with diethylamino. Sulfatrifluoride (DAST, 17μ, 0.13mmol) was added, then the ice bath was removed and stirred at room temperature for a further 15 minutes. After adding a few drops of methanol to inactivate the excess reagent, the reaction mixture was concentrated under reduced pressure, and the residue was extracted with ether-ethyl acetate (1: 1). After extraction, the extract was washed with water and saturated saline, dried over anhydrous sodium sulfate, and concentrated to obtain a crude product. Purify by column chromatography using silica gel (20 g), develop with toluene-ethyl acetate (3: 1), and add compound (3) (126 mg, 76 μmol, 96.8%) to the anomer of α / β = 7/18. Obtained as a mixture. (The production ratio was confirmed by high performance liquid chromatography and NMR spectrum).

[Properties of Compound (3)] ¹ H-NMR (500 MHz, CDCl ₃ ) δ 1.782 (3H, s, COC
H ₃ ), (1.885) 1.903 (3H, s, COC H ₃ ), 1.924 (1.9
48) (3H, s, COC H ₃ ), 2.036 (3H, s, COC H ₃ ), 5.180
(Dd, J = 52.8,5.8Hz, H-1a, β-anomer), 5.581 (d, J
= 56.2 Hz, H-1a, α-anomer).

Elemental analysis Analytical value C, 69.21; H, 6.29; F, 1.13 Calculated value (C ₉₅ H ₁₀₃ O ₂₄ F) C, 69.24; H, 6.30; F, 1.15% Reference Example 6 t-butyldiphenylsilyl O- (2 , 3-Di-O-benzyl-4,6-O-isopropylidene-α-D-galactopyranosyl)-(1 → 4) -O- (6-O-acetyl-2,3-di-O -Benzyl-α-D-galactopyranosyl)-(1 → 4) -O- (6-O-acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl)-(1
→ 4) -6-O-acetyl-2,3-di-O-benzyl-
β-D-galactopyranoside (13) stannous chloride (290mg, 1.53mmol), silver perchlorate (315m
g, 1.52 mmol), molecular sieves 4A powder (3 g), and the known compound (12) (57) in anhydrous ether (35 ml).
8mg, 0.73mmol, 1.1eq) and compound (8) (680mg, 0.6
6 mmol) was reacted at -10 to 10 ° C for 1.5 hours.

The crude product obtained by post-treatment in the same manner as in Reference Example 3 was purified by column chromatography using silica gel (150 g) and n-hexane-ethyl acetate-pyridine (70: 30: 1).
The compound (13) (920mg, 0.51mmol, 77.4
%) Was obtained.

[Compound properties (13)] [alpha ▲] _{^{24 D ▼ + 64.3 (c,}} 1.0, CHCl 3) 1 H-NMR (500MHz, CDCl 3) δ 1.128 [9H, s, -C (C H
₃ ) ₃ ], 1.326 [3H, s, C (C H ₃ ) ₂ ], 1.409 [3
_{H, s, C (C H} 3) 2 ], 1.888 (3H, s, COC H 3), 1.8
88 (3H, s, COC H ₃ ), 2.007 (3H, s, COC H ₃ ), 3.034
(1H, d, J = 11.9Hz, H-6a), 3.212 (1H, t, J = 6.7Hz, H-5
a), 3.241 (1H, dd, J = 9.8,2.7Hz, H-3a), 3.313 (1H,
d, J = 11.3Hz, H-6d), 3.633 (1H, dd, J = 9.8,7.3Hz, H-2
a), 3.72-4.96 (39H, m), 7.16-7.49 (46H, m, aromatic), 7.69-7.73 (4H, m, aromatic). ^{13 C-NMR (22.5MHz, CDCl} 3) δ 18.26 [C (C H _3)
2], 19.23 [- C (CH _{3) 3],} 20.70 [CO C H _3), 20.
_{86 (CO C H 3),} 27.09 [-C (C H _{3) 3],} 29.47 [C
( C H ₃ ) ₂ ], 97.96 (C-1a), 98.34 [ C (C
H ₃ ) ₂ ], 99.15, 99.42, 100.24 (C-1b, c, d], 169.70
(C = 0), 169.85 (C = 0), 170.23 (C = 0).

Elemental analysis Analytical value C, 69.91; H, 6.60 Calculated value (C ₁₀₅ H ₁₁₈ O ₂₄ Si) C, 70.37; H, 6.64% Reference Example 7 O- (2,3-di-O-benzyl-4,6- O-isopropylidene-α-D-galactopyranosyl)-(1 → 4) -O
-(6-O-Acetyl-2,3-di-O-benzyl-α-
D-galactopyranosyl)-(1 → 4) -O- (6-O
-Acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl)-(1 → 4) -6-O-acetyl-2,3
-Di-O-benzyl-D-galactopyranose (14) Silyl compound (13) (390 mg, 0.22 mmol) in tetrahydrofuran (6.6 ml) in the presence of glacial acetic acid (90 µ, 1.57 mmol) in 1M tetra n-butyl ammonium. Stir with fluoride (0.9 ml, 0.9 mmol) at room temperature for 3 days. Reference example 4
The same post-treatment was carried out and purified on silica gel (40 g). It was developed with toluene-ethyl acetate-pyridine (65:35, 1) to obtain the compound (14) (325 mg, 0.21 mmol, 96.1%).

[Properties of Compound (14)] ¹ H-NMR (500 MHz, CDCl ₃ ) δ 1.323 [3 H, s, C (C
H _{3) 2],} 1.403 _{[3H, s, C (C H} 3) 2 ], 1.935
(3H, s, COC H ₃ ), 1.963 (1.978) (3H, s, COC H ₃ ),
(2.031) 2.033 (3H, s , COC H 3), 5.266 (H-1a, α- anomer).

Elemental analysis Analytical value C, 68.31; H, 6.50 Calculated value (C ₈₉ H ₁₀₀ O ₂₄ ) C, 68.80; H, 6.49% Reference Example 8 O- (2,3-di-O-benzyl-4,6-O) -Isopropylidene-α-D-galactopyranosyl)-(1 → 4) -O
-(6-O-Acetyl-2,3-di-O-benzyl-α-
D-galactopyranosyl)-(1 → 4) -O- (6-O
-Acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl)-(1 → 4) -6-O-acetyl-2,3
-Di-O-benzyl-α-D-galactopyranosyl fluoride (4) According to Reference Example 5, hemiacetal (14) (300 mg, 0.19 m
mol) was reacted with diethylaminosulfur trifluoride (42μ, 0.32mmol) in anhydrous tetrahydrofuran (1.5ml) and the crude product was purified by column chromatography on silica gel (25g), toluene-ethyl acetate-pyridine (70 Expanded at 30: 1) Compound (4) (290
mg, 0.19 mmol, 96.5%) was obtained as an anomeric mixture of α / β = 8/17. (High performance liquid chromatography and ¹ H-
The production ratio is checked from the NMR spectrum).

[Compound (4) the nature] ^{1 H-NMR (500MHz, CDCl} 3) δ 1.325 [3 H, s, C (C H
₃ ) ₂ ], 1.408 [3H, s, C (CH ₃ ) ₂ ], 1.923 (1.94
6) (3H, s, COCH ₃ ), (1.965) 1.982 (3H, s, COCH ₃ ),
5.177 (dd, J = 52.8, 6.1Hz, H-1a, β-anomer), 5.585
(D, J = 55.5 Hz, H-1a, α-anomer).

Elemental analysis Analytical value C, 68.99; H, 6.45; F, 1.22 Calculated value (C ₈₉ H ₉₉ O ₂₃ F) C, 68.71; H, 6.41; F, 1.22% Reference Example 9 t-butyldiphenylsilyl O- (2 , 3-Di-O-benzyl-α-D-galactopyranosyl)-(1 → 4)-
O- (6-O-acetyl-2,3-di-O-benzyl-α
-D-galactopyranosyl)-(1 → 4) -O- (6-
O-acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl)-(1 → 4) -6-O-acetyl-2,
3-di-O-benzyl-β-D-galactopyranoside (1
5) 80% of isopropylidene derivative (13) (511mg, 0.29mmol)
Heated in hydrous acetic acid (8 ml) at 60 ° C. for 30 minutes. After concentration under reduced pressure, the residue was further azeotropically distilled with toluene to remove traces of acetic acid and water, and then subjected to column chromatography using silica gel (75 g).

It was developed with n-hexane-ethyl acetate (1: 1) to obtain compound (15) (457 mg, 0.26 mmol, 91.5%).

[Properties of compound (15)] [α ▲]²⁶ _D▼ + 63.4 (c, 1.0, CHCl₃)¹ H-NMR (500MHz, CDCl₃) Δ 1.126 [9H, s, -C (CH _Three)
_Three], 1.895 (6H, s, COCH ₃ x2), 1.948 (3H, s, COC
H _Three), 4.554 (1H, d, J = 7.OHz, H-1a), 4.960 (2H, d, J
= 3.4Hz) & 4.984 (1H, d, J = 3.4Hz) (H-1b, c, d).¹³ C-NMR (22.5MHz, CDCl₃) Δ 19.23 (-CMe₃), 20.6
4 (COCH₃), 20.81 (COCH₃), 27.04 [C (C
H₃)_Three], 97.96 (¹J_CH 158.7Hz, C-1a), 99.15,99.37,
99.80 (¹J_CH(Average) 173.3Hz, C-1b, c, d), 169.80 (C
= O), 169.91 (C = O), 170.23 (C = O).

Elemental analysis Analytical value C, 69.16; H, 6.54 Calculated value (C ₁₀₂ H ₁₁₄ O ₂₄ Si.H ₂ O) C, 69.21; H, 6.49% Reference Example 10 t-Butyldiphenylsilyl O- (6-O-acetyl -2,3-Di-O-benzyl-α-D-galactopyranosyl)-(1 → 4) -O- (6-O-acetyl-2,3-di-O-benzyl-α-D- Galactopyranosyl)-(1
→ 4) -O- (6-O-acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl)-(1 → 4) -6
-O-Acetyl-2,3-di-O-benzyl-β-D-galactopyranoside (5) Diol (15) (415 mg, 0.24 mmol) was dissolved in anhydrous pyridine (2.5 ml), and the mixture was placed on an ice bath. Acetyl chloride (21μ, 0.30mmol, 1.25eq) was added while cooling with, and the mixture was stirred for 2.5 hours. Further, acetyl chloride (5 μ, 0.07 mmol) was added, and stirring was continued at 5 ° to 15 ° for 1.5 hours. The reaction was stopped by adding water and post-treatment was carried out as in Reference Example 2, and the crude product was purified by column chromatography using silica gel (70 g) and toluene-ethyl acetate (7: 3). First, by-product pentaacetate (19 mg, 0.01 mmol, 4.4%) {[α ▲] ²⁵ _D ▼ +56.5 (c, 0.8, CHCl ₃ ), ¹ H-NMR (500 MHz, CDCl ₃ ) δ 1.126 [9H _{, s, -C (C H 3} )
₃ ], 1.896 (3H, s, COC H ₃ ), 1.915 (3H, s, COC
H ₃ ), 1.935 (3H, s, COCH ₃ ), 1.959 (3H, s, COC
H ₃ ), 2.043 (3H, s, COC H ₃ ), 5.531 (1H, m, H-4
a), Analytical value C, 69.70; H, 6.55 Calculated value C, 69.34; H, 6.48
%} Was obtained. Next, the target product (5) (395 mg, 0.22 mmol, 93.
0%) was obtained.

[Compound (5) the nature] [alpha ▲] _{^{26 D ▼ + 57.32 (c,}} 0.64, CHCl 3), 1 H-NMR (500MHz, CDCl 3) δ 1.121 [9H, s, -C (C H 3 )
₃ ], 1.893 (3H, s, COC H ₃ ), 1.896 (3H, s, COC
H ₃ ), 1.910 (3H, s, COC H ₃ ), 1.941 (3H, s, COC
_{H 3), 3.219 (1H,} t, J = 6.7Hz, H-5a), 3.251 (1H, dd,
J = 10.1,2.7Hz, H-3a), 3.637 (1H, dd, J = 10.1,7.3Hz, H
-2a), 4.549 (1H, d, J = 7.3Hz, H-1a), 4.948 (3H, m, H-
1b, c, d). ¹³ C-NMR (22.5 MHz, CDCl ₃ ) δ 19.23 ( C Me ₃ ), 20.81
(CO C H _3), 27.04 [C (C H _{3) 3],} 97.96 (C-1a),
99.31,99.58 (C-1b, c, d), 169.80 (C = O), 169.91
(C = O), 170.18 (C = O).

Elemental analysis Analytical value C, 68.81; H, 6.48 Calculated value (C ₁₀₄ H ₁₁₆ O ₂₅ Si · H ₂ O) C, 68.93; H, 6.45% Example 1 t-butyldiphenylsilyl O- (2,3-di -O-benzyl-4,6-O-isopropylidene-α-D-galactopyranosyl)-(1 → 4) -O- (6-O-acetyl-2,3-di-O-benzyl-α -D-galactopyranosyl)-(1 → 4) -O- (6-O-acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl)-(1
→ 4) -O- (6-O-acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl)-(1 → 4) -O
-(6-O-Acetyl-2,3-di-O-benzyl-α-
D-galactopyranosyl)-(1 → 4) -O- (6-O
-Acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl)-(1 → 4) -O- (6-O-acetyl-2,3-di-O-benzyl-α- D-galactopyranosyl)-(1 → 4) -6-O-acetyl-2,3-di-O-
Benzyl-β-D-galactopyranoside (16) Stannous chloride (45 mg, 0.24 mmol), silver perchlorate (50 mg,
0.24 mmol) and molecular sieves 4A powder (400 mg) were used, and according to Reference Example 3, fluoride (4) (130 mg, 84 μmol, 1.4 equivalent) and sugar acceptor (5) (107 mg, 60 μmol) in anhydrous ether (4 ml). And were reacted at -10 ° to 10 ° C for 3.5 hours. Then, the crude product obtained by the same post-treatment was subjected to column chromatography on silica gel (40 g) and eluted with toluene-ethyl acetate-pyridine (70: 30: 1) to obtain a compound (16) ( 135 mg) was used as the first fraction, and then a second fraction containing the deisopropylidenediated diol (17) (38 mg) as the main component was obtained.

The following treatments were carried out without further purification.

Example 2 t-Butyldiphenylsilyl O- (2,3-di-O-benzyl-α-D-galactopyranosyl)-(1 → 4)-
O- (6-O-acetyl-2,3-di-O-benzyl-α
-D-galactopyranosyl)-(1 → 4) -O- (6-
O-acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl)-(1 → 4) -O- (6-O-acetyl-2,3-di-O-benzyl-α -D-galactopyranosyl)-(1 → 4) -O- (6-O-acetyl-2,3-)
Di-O-benzyl-α-D-galactopyranosyl)-
(1 → 4) -O- (6-O-acetyl-2,3-di-O-
Benzyl-α-D-galactopyranosyl)-(1 → 4)
-O- (6-O-acetyl-2,3-di-O-benzyl-
α-D-galactopyranosyl)-(1 → 4) -6-O-
Acetyl-2,3-di-O-benzyl-β-D-galactopyranoside (17) The first and second fractions of the product of the previous reaction were 2 ml and 1 ml, respectively.
In 80% hydrous acetic acid at 60 ° C. for 30 minutes. After concentration under reduced pressure, the former was subjected to silica gel (20 g) column chromatography and the latter was subjected to thin layer chromatography (Kieselge).
Compounds (17) (101 mg and 21 mg, developed with toluene-ethyl acetate (7: 3)
A cumulative yield of 62.2% from (5) was obtained.

[Compound (17) properties] [alpha ▲] _{^{24 D ▼ + 61.9 (c,}} 0.7, CHCl 3) 1 H-NMR (500MHz, CDCl 3) δ 1.114 [9H, s, -C (C H 3)
₃ ], 1.878 (9H, s, COC H ₃ x3), 1.886 (3H, s, COC
H ₃ ), 1.902 (9H, s, COC H ₃ x3), 3.214 (1H, br.t, H-
5a), 3.246 (1H, dd, J = 9.7 2.75Hz, H-3a), 3.382 (1
H, m, H-6h), 3.435 (1H, m, H-6h), 3.632 (1H, dd, J = 9.
77,7.33Hz, H-2a), 4.534 (1H, d, J = 7.33Hz, H-1a). ¹³ C-NMR (22.5 MHz, CDCl ₃ ) δ 19.23 ( -C Me ₃ ), 20.8
_{1 (CO C H 3),} 27.09 [-C (C H _{3) 3],} 98.07 ⁽¹ J _CH
159.9Hz, C-1a), 99.26,99.75 ( ¹ J _CH 170.OHz, C-1b ~
h), 169.75 (C = O), 170.23 (C = O), 170.67
(C = O).

Elemental analysis Analytical value C, 69.54; H, 6.45 Calculated value (C ₁₉₀ H ₂₁₀ O ₄₈ Si) C, 69.37; H, 6.43% Example 3 t-butyldiphenylsilyl O- (6-O-acetyl-2,3 -Di-O-benzyl-α-D-galactopyranosyl)-(1 → 4) -O- (6-O-acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl) )-(1
→ 4) -O- (6-O-acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl)-(1 → 4) -O
-(6-O-Acetyl-2,3-di-O-benzyl-α-
D-galactopyranosyl)-(1 → 4) -O- (6-O
-Acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl)-(1 → 4) -O- (6-O-acetyl-2,3-di-O-benzyl-α- D-galactopyranosyl)-(1 → 4) -O- (6-O-acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl)-(1
→ 4) -6-O-acetyl-2,3-di-O-benzyl-
β-D-galactopyranoside (18) Diol (17) (80 mg, 24 μmol) was added to anhydrous pyridine (0.6
ml) and acetyl chloride (4.5μ, 63μmol) and 0 ° -10
Stirred at 4 ° C for 4 hours. After the post-treatment as in Reference Example 2, the crude product was purified by column chromatography on silica gel (15 g) and eluted with toluene-ethyl acetate (3: 1) to give compound (18) (66 mg, 81.5%). Obtained. (At this time, almost no corresponding nonaacetyl derivative was obtained).

[Compound (18) properties] [alpha ▲] _{^{22 D ▼ + 53.8 (c,}} 0.7, CHCl 3) 1 H-NMR (500MHz, CDCl 3) δ 1.115 [9H, s, -C (C H 3)
₃ ], 1.868 (9H, s, COC H ₃ ), 1.879 (3H, s, COC
H ₃ ), 1.885 (3H, s, COC H ₃ ), 1.896 (3H, s, COC
H ₃ ), 1.899 (3H, s, COC H ₃ ), 1.901 (3H, s, COC
H ₃ ), 1.904 (3H, s, COC H ₃ ), 1.954 (3H, s, COC
H ₃ ), 3.227 (1H, br.t, H-5a), 3.246 (1H, dd, J = 9.7
7,2.75Hz, H-3a), 3.631 (1H, dd, J = 9.77,7.02Hz, H-2
a), 4.544 (1H, d, J = 7.02Hz, H-1a). ¹³ C-NMR (22.5 MHz, CDCl ₃ ) δ 19.18 ( -C Me ₃ ), 20.7
0 (CO C H ₃ ), 26.98 [C ( C H ₃ ) ₃ ], 97.79 (C-1
a), 99.15 (C-1b to h), 169.70 (C = O), 169.80
(C = O), 170.13 (C = O).

Elemental analysis analysis C, 68.47; H, 6.36 Calculated _{(C 192 H 212 O 49 Si} · H 2 O) C, 68.84; H, 6.44% Example 4 t-Butyl-diphenyl-O- (6-O- acetyl - 2,3,
4-Tri-O-benzyl-α-D-galactopyranosyl)-(1 → 4) -O- (6-O-acetyl-2,3-di-O-benzyl-α-D-galactopyrano Sill)-(1
→ 4) -O- (6-O-acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl)-(1 → 4) -O
-(6-O-Acetyl-2,3-di-O-benzyl-α-
D-galactopyranosyl)-(1 → 4) -O- (6-O
-Acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl)-(1 → 4) -O- (6-O-acetyl-2,3-di-O-benzyl-α- D-galactopyranosyl)-(1 → 4) -O- (6-O-acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl)-(1
→ 4) -O- (6-O-acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl)-(1 → 4) -O
-(6-O-Acetyl-2,3-di-O-benzyl-α-
D-galactopyranosyl)-(1 → 4) -O- (6-O
-Acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl)-(1 → 4) -O- (6-O-acetyl-2,3-di-O-benzyl-α- D-galactopyranosyl)-(1 → 4) -O-6-O-acetyl-2,3-di-
O-benzyl-β-D-galactopyranoside (19) stannous chloride (23 mg, 0.12 mmol), silver perchlorate (26 mg,
0.13 mmol) and molecular sieves 4A powder (400 mg), and in the same manner as in Reference Example 3, fluoride (3) (75 mg, 46 μmo)
l, 1.5 equivalents) and the sugar acceptor (18) (100 mg, 30 μmol) were reacted in a mixed solvent of anhydrous ether (3.5 ml) -anhydrous toluene (1.5 ml) at −10 ° C. to room temperature overnight. The insoluble material was filtered through Celite and washed with chloroform. The filtrate was further diluted with chloroform, washed with water and saturated saline, and then dried over anhydrous sodium sulfate. The crude product remaining after concentration under reduced pressure was purified by column chromatography using silica gel (25 g). Eluted with toluene-ethyl acetate (3: 1) to give a compound (1
9) (117 mg) was obtained. This was further purified by thin layer chromatography (Kieselgel 60, 0.5 mm, 20 × 20 cm, 3 sheets) to obtain compound (19) (95 mg, 19 μmol, 63.5%).

[Properties of compound (19)] [α ▲]^{twenty two} _D▼ + 55.8 (c, 0.6, CHCl₃)¹ H-NMR (500MHz, CDCl₃) Δ 1.115 [9H, s, C (CH _Three)
_Three], 1.782 (3H, s, COCH _Three), 1.853 (3H, s, COC
H _Three), 1.881 (3H, s, COCH _Three), 1.887 (9H, br.s, COC H
₃ x3), 1.889 (9H, br.s, COCH ₃ x3), 1.901 (3H, s, CO
CH _Three), 1.908 (3H, s, COCH _Three), 1.958 (3H, s, COCH
_Three), 3.210 (1H, br.t, H-5a), 3.244 (1H, dd, J = 9.77,
2.44Hz, H-3a), 3.629 (1H, dd, J = 9.77,7.02Hz, H-2
a), 4.558 (1H, d, J = 7.02Hz, H-1a)¹³ C-NMR (22.5MHz, CDCl₃), Δ 19.29 (CMe₃), 20.8
1 (COCH₃), 27.09 [C (CH₃)_Three], 98.07 (C-1
a), 99.26,99.96 (C-1b to l), 169.75 (C = O), 169.91 (C = O), elemental analysis analysis value C, 68.92; H, 6.29 calculated value (C₂₈₇H₃₁₄O₇₃Si; H₂O) C, 69.25; H, 6.36 Example 5 t-Butyldiphenylsilyl O- (2,3-di-O-be
And -α-D-galactopyranosyl- (1 → 4) -O
-(2,3-Di-O-benzyl-α-D-galactopyrano
Sil)-(1 → 4) -O- (2,3-di-O-benzyl-
α-D-galactopyranosyl)-(1 → 4) -O- (2,
3-di-O-benzyl-α-D-galactopyranosyl-
(1 → 4) -O- (2,3-di-O-benzyl-α-D-
Galactopyranosyl- (1 → 4) -O- (2,3-di-O
-Benzyl-α-D-galactopyranosyl- (1 → 4)
-O- (2,3-di-O-benzyl-α-D-galactopi
Lanosyl- (1 → 4) -O- (2,3-di-O-benzyl
Le-α-D-galactopyranosyl- (1 → 4) -O-
(2,3-di-O-benzyl-α-D-galactopyranosi
Ru- (1 → 4) -O- (2,3-di-O-benzyl-α-
D-galactopyranosyl- (1 → 4) -O- (2,3-di
-O-benzyl-α-D-galactopyranosyl- (1 →
4) -O-2,3-di-O-benzyl-β-galactopyra
Noside (20) Compound (19) (55 mg, 11 μmol) was added to anhydrous tetrahydrofuran.
Dissolve in orchid (0.5 ml) and add 0.1N sodium methoxide
Add SID methanol solution (1 ml) and stir overnight at room temperature
did. The reaction mixture was diluted with methanol and amberlyst 15
After neutralizing with, it was filtered and concentrated. Thin the resulting crude product
Layer chromatography (Kieselgel 60, 0.5mm, 20x20c
m, 3 sheets). Toluene-chloroform-
Develop with ethyl acetate (3: 2: 5) and compound (20) (40mg,
9 μmol, 81%) was obtained.

[Properties of Compound (20)] [alpha ▲] _{^{22 D ▼ + 79.11 (c,}} 0.5, CHCl 3) 1 H-NMR (500MHz, CDCl 2) δ 1.091 _{[9H, s, C (C H} 3)
₃ ], 4.545 (1H, d, J = 7.33Hz, c-1a) Example 6 t-butyldiphenylsilyl O- (2,3,4-tri-O
-Benzyl-α-D-galactopyranosyluronic acid)-
(1 → 4) -O- (2,3-di-O-benzyl-α-D-
Galactopyranosyluronic acid)-(1 → 4) -O- (2,
3-di-O-benzyl-α-D-galactopyranosyluronic acid)-(1 → 4) -O- (2,3-di-O-benzyl-α-D-galactopyranosyluronic acid) -(1 → 4)
-O- (2,3-di-O-benzyl-α-D-galactopyranosyluronic acid)-(1 → 4) -O- (2,3-di-O
-Benzyl-α-D-galactopyranosyluronic acid)-
(1 → 4) -O- (2,3-di-O-benzyl-α-D-
Galactopyranosyluronic acid)-(1 → 4) -O- (2,
3-di-O-benzyl-α-D-galactopyranosyluronic acid)-(1 → 4) -O- (2,3-di-O-benzyl-α-D-galactopyranosyluronic acid) -(1 → 4)
-O- (2,3-di-O-benzyl-α-D-galactopyranosyluronic acid)-(1 → 4) -O- (2,3-di-O
-Benzyl-α-D-galactopyranosyluronic acid)-
(1 → 4) -2,3-di-O-benzyl-β-D-galactopyranosideuronic acid (31) Oxalyl chloride (53μ, 608μmol) in anhydrous dichloromethane (1.3ml) was added to dry ice-acetone. The mixture was cooled on the bath and stirred under a stream of argon, to which anhydrous dimethyl sulfoxide (100 μ, 1.4 mmol) was added and stirred for 15 minutes.

Next, a solution of compound (20) (40 mg, 9.0 μmol) in anhydrous dichloromethane (1.5 ml) was added and stirred for 15 minutes, then N, N-diisopropylethylamine (450 μ, 2.6 mmol) was added and stirred for 5 minutes, and then cooled. The bath was removed and stirring was continued for another 15 minutes. The reaction solution was diluted with chloroform (30 ml), washed with diluted hydrochloric acid (containing 2.6 mmol of hydrochloric acid), water and saturated saline, and then dried over anhydrous sodium sulfate. After concentration under reduced pressure, the residue was dissolved in t-butyl alcohol (2.5 ml), 2-methyl-2-butene (0.6 ml) and sodium chlorite (105 ml).
mg, 1.2mmol) and monosodium phosphate dihydrate (105m
g, 0.67 mmol) was dissolved in water (1.1 ml), added to the above and stirred at room temperature overnight. The reaction solution was concentrated under reduced pressure, water was added to the residue, and the mixture was extracted with n-hexane to remove soluble components.
Subsequently, dilute hydrochloric acid was added dropwise to the aqueous layer to make it acidic (pH 1-2) and extracted with ethyl acetate. The extract was washed with water and saturated saline and then dried over anhydrous sodium sulfate.

The crude product obtained by distilling off the solvent was purified by thin layer chromatography (Kieselgel 0.5 mm, 20 × 20 cm, 2 sheets).
Develop with chloroform-acetone-acetic acid (9: 1: 1),
Corresponding fractions were collected and extracted with chloroform-methanol-acetic acid (8: 1: 1). The solvent was distilled off and the remaining extract was dissolved in ethyl acetate, washed with diluted hydrochloric acid, water and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give compound (21) (20.6 mg, 49.6%). ) Got.

[Properties of Compound (21)] [α ▲] ²² _D ▼ + 141.1 (c, 0.4, 95: 5 CHCl ₃ : MeOH) For confirmation of the structure, a part of the corresponding methyl ester was prepared by using diazomethane. ^{1 H-NMR (500MHz, CDCl} 3) δ 1.170 _{[9H, s, C (C H} 3)
₃ ], 3.087 (3H, s, OC H ₃ ), 3.194 (3H, s, OC H ₃ ),
3.199 (6H, br.s, OC H ₃ x2), 3.204 (6H, br.s, OC H ₃ x
2), 3.208 (3H, s, OC H ₃ ), 3.237 (3H, s, OC H ₃ ),
3.253 (3H, s, OC H 3), 3.365 (3H, s, OC H 3), 3.443
Hydrolysis of (3H, s, OC H ₃ ), 3.449 (3H, s, OC H ₃ ) methyl ester (regeneration of compound (21)) Methyl ester (14.5 mg) with LiI (45 mg) in anhydrous pyridine (6 ml) The mixture was heated under reflux for 30 hours under a medium argon flow. After cooling, the mixture was concentrated under reduced pressure, the residue was dissolved in water and acidified with diluted hydrochloric acid. This was extracted with ethyl acetate, washed with water and saturated brine, and dried over anhydrous sodium sulfate. The crude product obtained by distilling off the solvent was purified on a thin layer chromatography plate (20 × 20 cm) having a thickness of 0.5 mm. CHCl ₃
It was developed with -acetone-AcOH (9: 1: 1) and the corresponding fractions were collected and extracted. Since the extract contains some inorganic substances, it is separated into ethyl acetate and diluted hydrochloric acid, the organic layer is washed with water, dried and concentrated as above to obtain compound (21) (9.3 mg, 66.
5%).

Example 7 O- (2,3,4-tri-O-benzyl-α-D-galactobiranosyluronic acid)-(1 → 4) -O- (2,3-di-
O-benzyl-α-D-galactopyranosyluronic acid)
-(1 → 4) -O- (2,3-di-O-benzyl-α-D
-Galactopyranosyluronic acid)-(1 → 4) -O-
(2,3-di-O-benzyl-α-D-galactopyranosyluronic acid)-(1 → 4) -O- (2,3-di-O-benzyl-α-D-galactopyranosyl Uronic acid)-(1 →
4) -O- (2,3-di-O-benzyl-α-D-galactopyranosyluronic acid)-(1 → 4) -O- (2,3-di-O-benzyl-α-D -Galactopyranosyluronic acid)-(1 → 4) -O- (2,3-di-O-benzyl-α
-D-galactopyranosyluronic acid)-(1 → 4) -O
-(2,3-Di-O-benzyl-α-D-galactopyranosyluronic acid)-(1 → 4) -O- (2,3-di-O-benzyl-α-D-galactopyrano Siluronic acid)-(1
→ 4) -O- (2,3-di-O-benzyl-α-D-galactopyranosyluronic acid)-(1 → 4) -2,3-di-O
-Benzyl-D-galactopyranuronic acid (22) Compound (21) (6 mg, 1.3 μmol) in anhydrous tetrahydrofuran (0.5 ml) glacial acetic acid (6.5 μ, 114 μmol) and IN
4 with room temperature tetra-n-butylammonium fluoride-tetrahydrofuran solution (65μ, 65μmol)
It was stirred for a day. The reaction mixture was diluted with water, diluted with dilute hydrochloric acid to make it acidic (pH 1-2), and extracted with ethyl acetate. The extract was washed with water and saturated saline and dried over anhydrous sodium sulfate. The crude product obtained by distilling off the solvent was purified by a gel filtration column (Toyopearl HW-40SF, 30 ml), and chloroform-methanol-acetic acid (9: 1: 1) was used for development. The fractions were separated while monitoring by thin layer chromatography to obtain the fraction of the target product (22). Since this fraction contained inorganic salts, it was redissolved in ethyl acetate, washed with dilute hydrochloric acid, water and saturated brine, dried over anhydrous sodium sulfate and concentrated to give the desired product (22) (5.2m
g, 1.2 μmol 91%) was obtained.

Example 8 O- (α-D-galactopyranosyluronic acid)-(1 →
4) -O- (α-D-galactopyranosyluronic acid)-
(1 → 4) -O- (α-D-galactopyranosyluronic acid)-(1 → 4) -O- (α-D-galactopyranosyluronic acid)-(1 → 4) -O- (Α-D-galactopyranosyluronic acid)-(1 → 4) -O- (α-D-galactopyranosyluronic acid)-(1 → 4) -O- (α-D-
Galactopyranosyluronic acid)-(1 → 4) -O- (α
-D-galactopyranosyluronic acid)-(1 → 4) -O
-(Α-D-galactopyranosyluronic acid)-(1 →
4) -O- (α-D-galactopyranosyluronic acid)-
80% of (1 → 4) -O- (α-D-galactopyranosyluronic acid)-(1 → 4) -α-D-galactopyranuronic acid (2) compound (22) (4.2 mg) In water-containing methanol (3 ml)
10% Palladium on carbon (3 mg) was added, and the mixture was stirred under a hydrogen stream for 4 days at room temperature. After filtering through Celite,
Chromatography was performed using Sephadex LH-20 (12 ml) to obtain a fraction containing galacturonic acid. Next, this was subjected to ion exchange chromatography on a Mono Q (HR5 / 5) column using a Pharmacia FPLC system. Elute with a linear gradient (0.2M–1M) of NH ₄ · HCO ₃ buffer, and use the m-hydroxydiphenyl method [N. Blumenkrantz and GA] for each fraction.
sboe-Hansou, Anal. Biochem, 54 484-489 (1973)]. Fractions containing compound (2) were collected and concentrated to obtain compound (2) (1.5 mg).

[Properties of Compound (2)] [α ▲] ²² _D ▼ + 80.8 (c, 0.1, H ₂ O) ¹ H-NMR [D ₂ O, internal standard t-BuOH (1.23), 80 ° C.]: δ
3.49 [dd, J = 7.6, 10, 1 Hz, H-2a (β-anomer)], 3.7
6 (12H, brd, H-2b to l, H-3a), 3.90 (1H, dd, J = 3.7,10.
1 Hz, H-3l), 3.98 (11H, brd, H-3b to 1), 4.45 (11H,
brd, H-4b to 1), 4.58 [d, J = 7.6Hz, H-1a (β-anomer)], 4.77 (11H brs, H-5b to 1), 5.10 (11H, brs, H-
1b-1), 5.31 [brd, H-1a (α-anomer)].

Reference Example 11 t-Butyldiphenylsilyl O- (2,3-di-O-benzyl-4,6-O-isopropylidene-α-D-galactopyranosyl)-(1 → 4) -O- (6 -O-acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl)-(1 → 4) -O- (6-O-acetyl-2,3-di-O-benzyl- α-D-galactopyranosyl)-(1
→ 4) -O- (6-O-acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl)-(1 → 4) -O
-(6-O-Acetyl-2,3-di-O-benzyl-α-
D-galactopyranosyl)-(1 → 4) -6-O-acetyl-2,3-di-O-benzyl-β-D-galactopyranoside (23) Stannous chloride according to Reference Example 3 (45mg, 0.24mmol), silver perchlorate (50mg, 0.24mmol), molecular sieves 4A
Reaction of fluoride (12) (90 mg, 114 μmol, 1.3 equivalents) with sugar acceptor (5) (155 mg, 86 μmol) in anhydrous ether (5.5 ml) at −10 ° to 15 ° C for 3 hours using powder (0.5 g) Let After similar post treatment, the crude product was treated with silica gel (40
g) was purified by column chromatography and eluted with toluene-ethyl acetate-pyridine (80: 20: 1), and the compound (23) (155 mg) containing some impurities, and toluene-ethyl acetate (1: Elute with 1) and diol (24) (33m
A fraction based on g) was obtained.

[Properties of Compound (23)] ¹ H-NMR [90 MHz, CDCl ₃ ) δ1.12 [9 H, s, C (C
H _{3) 3],} 1.32 _{[3H, s, C (C H} 3) 2 ], 1.40 [3H,
s, C (C H ₃ ) ₂ ], 1.89 (6H, brs, COC H ₃ x2), 1.92
(3H, s, COC H ₃ ), 1.94 (3H, s, COC H ₃ ), 1.96 (3H, s,
COC H ₃₎ Reference Example 12 t-butyldiphenylsilyl O-(2,3-di--O- benzyl-.alpha.-D-galactopyranosyl) - (1 → 4) -
O- (6-O-acetyl-2,3-di-O-benzyl-α
-D-galactopyranosyl)-(1 → 4) -O- (6-
O-acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl)-(1 → 4) -O- (6-O-acetyl-2,3-di-O-benzyl-α -D-galactopyranosyl)-(1 → 4) -O- (6-O-acetyl-2,3-)
Di-O-benzyl-α-D-galactopyranosyl)-
(1 → 4) -6-O-acetyl-2,3-di-O-benzyl-β-D-galactopyranoside (24) The two fractions obtained in the synthesis of compound (23) were combined and Heated in 60% hydrous acetic acid (4 ml) at 60 ° C. for 30 minutes. The crude product remaining after concentration was purified by column chromatography on silica gel (35 g). Compound (24) (162 mg, 64 μmo) eluted with toluene-ethyl acetate (7: 3).
l, 74.4%) was obtained from compound (5).

[Properties of Compound (24)] [α ▲] ²³ _D ▼ +58.7 (c, 0.5, CHCl ₃ ) ¹ H-NMR [500 MHz, CDCl ₃ ) δ1.115 [9 H, s, C (C
H ₃ ) ₃ ], 1.882 [9H, br.s, COC H ₃ x3), 1.914 (3H,
s, COC H ₃ ), 1.962 (3H, s, COC H ₃ ), 3.216 (1H, br.
s, H-5a), 3.248 (1H, dd, J = 9.77,2.75Hz, H-3a), 3.39
2 (1H, m, H-6f), 3.441 (1H, dd, J = 11.59,3.66Hz, H-6
f), 3.634 (1H, dd, J = 9.77,7.32Hz, H-2a), 4.547 (1
H, d, J = 7.32Hz, H-1a) ¹³ C-NMR (22.5MHz, CDCl ₃ ) δ 19.18 ( C Me ₃ ), 20.75 (C
O C H _3), 26.98 [C (C H _{3) 3],} 97.90 ⁽¹ J _CH 159.9H
z), 99.20,99.69 ( ¹ J _CH 170.9Hz, C-1b to f), 169.58
(C = O), 169.70 (C = O), 169.85 (C = O), 17
0.18 (C = O) Elemental analysis Analytical value C, 69.92; H, 6.53 Calculated value (C ₁₄₆ H ₁₆₂ O ₃₆ Si) C, 69.56; H, 6.48% Reference example 13 t-Butylphenylsilyl O- (6-O -Acetyl-
2,3-di-O-benzyl-α-D-galactorapyranosyl)-(1 → 4) -O- (6-O-acetyl-2,3-di-O-benzyl-α-D-galacto Pyranosyl)-(1
→ 4) -O- (6-O-acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl) -α-D-galactopyranosyl)-(1 → 4)- O- (6-O-acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl)-(1 → 4) -O- (6-O-acetyl-2,3-
Di-O-benzyl-α-D-galactopyranosyl)-
(1 → 4) -6-O-Acetyl-2,3-di-O-benzyl-β-D-galactopyranoside (25) Diol (24) (145 mg, 58 μmol) was added to anhydrous pyridine (1.
Acetyl chloride (5.3μ, 75μmol) in 5ml) and stirred on ice-water bath for 2 hours. Acetyl chloride (6μ, 84μmo
l) was added and stirred for 3 hours. The reaction was stopped by adding water and the crude product was treated with silica gel (30 g) in the same manner as in Reference Example 2 to carry out post-treatment.
Was purified by column chromatography. Toluene-
Elute with ethyl acetate (7: 3) to elute compound (25) (123 mg, 48
μmol, 83.4%) was obtained.

[Compound (25) properties] [alpha ▲] _{^{24 D ▼ + 53.8 (c,}} 0.5, CHCl 3) 1 H-NMR [500MHz, CDCl ₂₎ δ 1.116 _{[9H, s, C (C H} 3)
₃ ], 1.875 [3H, s, COC H ₃ ], 1.881 (3H, s, COC
_{H 3), 1.887 (3H,} s, COC H 3), 1.899 (3H, s, COC
H ₃ ), 1.917 (3H, s, COC H ₃ ), 1.963 (3H, s, COC
H ₃ ), 3.215 (1H, br.t, H-5a), 3.247 (1H, dd, J = 9.77,
2.75Hz, H-3a), 3.633 (1H, dd, J = 9.77,7.32Hz, H-2
a), 4.545 (1H, dd, J = 7.32Hz, H-1a), ¹³ C-NMR (22.5
_{MHz, CDCl 3) δ19.23 (C} Me 3), 20.70 (CO C H 3), 27.0
_{4 (C (C H 3)} 3 ], 97.96 (C-1a), 99.26 (C-1b~
f), 169.63 (C = O), 169.75 (C = O), 169.85
(C = O), 170.18 (C = O) Elemental analysis Analytical value C, 69.23; H, 6.45 Calculated value (C ₁₄₈ H ₁₆₄ O ₃₇ Si) C, 69.36; H, 6.45% Reference Example 14 t-Butyldiphenylsilyl O- (2,3-di-O-benzyl-α-D-galactopyranosyl)-(1 → 4)-
O- (6-O-acetyl-2,3-di-O-benzyl-α
-D-galactopyranosyl)-(1 → 4) -O- (6-
O-acetyl-2,3-di-O-benzyl-α-D-galactopyranosyl)-(1 → 4) -O- (6-O-acetyl-2,3-di-O-benzyl-α -D-galactopyranosyl)-(1 → 4) -O- (6-O-acetyl-2,3-)
Di-O-benzyl-α-D-galactopyranosyl)-
(1 → 4) -O- (6-O-acetyl-2,3-di-O-
Benzyl-α-D-galactopyranosyl)-(1 → 4)
-O- (6-O-acetyl-2,3-di-O-benzyl-
α-D-galactopyranosyl)-(1 → 4) -6-O-
Acetyl-2,3-di-O-benzyl β-D-galactopyranoside (17) Stannous chloride (22 mg, 116 μmol), silver perchlorate (24 mg,
116 μmol), molecular sieves 4A powder (250 mg) in anhydrous ether (3 ml) fluoride (12) (42 mg, 5
3μmol, 1.35eq) and compound (25) (100mg, 40μmol)
And were reacted in an ice-methanol bath (-10 to 10 ° C) for 3 hours. After the post-treatment as in Reference Example 3, the crude product was purified by column chromatography on silica gel (25 g) and eluted with toluene-ethyl acetate-pyridine (80: 20: 1) to give compound (16) as the main component. (69 mg), then the raw material (25) as the main component (16 mg), and toluene-ethyl acetate (3: 2) to elute the compound (17)
A fraction (39 mg) containing as a main component was obtained.

80% water-containing acetic acid (2m
l) and a few drops of tetrahydrofuran are added and dissolved at 60 ° C.
Heated for 30 minutes. After concentration under reduced pressure, the residue is silica gel (25 g)
Was purified by column chromatography. Toluene-
Elute with ethyl acetate (7: 3) to elute compound (17) (90 mg, 27
μmol, 70% from (25)). This is the same as the octasaccharide compound obtained by the condensation of Compounds (4) and (5) above, and the identity is ¹ H-NMR (500 MHz, CDCl ₃ ), ¹³ C-NMR.
(22.5MHz, CDCl ₃₎ was confirmed by.

〔The invention's effect〕

According to the present invention, a plant cell wall constituent fragment oligogalacturonic acid having high activity as an elicitor for inducing the production of antibacterial substance / phytoalexin can be precisely synthesized in high yield.

Claims (1)

[Claims] 1. A compound (4) represented by the formula (4) is condensed with a compound (5) represented by the formula (5) to obtain an octasaccharide compound (16), which is deisopropylidated. After preparing the compound (17), the 6-position is selectively acetylated to give 8
A sugar sugar acceptor (18) is prepared, and this is condensed with a tetrasaccharide sugar donor (3) represented by the formula (3) to give a disaccharide compound (1
9), which is deacetylated to give compound (20), which is then oxidized to lead to carboxylic acid (21) and the protecting group is eliminated, resulting in the 12 sugar represented by formula (2). For producing a soluble oligogalacturonic acid (2).