JP7723394B2 - Plant-derived organic molecule catalyst - Google Patents

Description

The present invention relates to a plant-derived organic molecular catalyst and a method for producing the same.

The compound represented by formula (Z) (hereinafter also referred to as "compound (Z)") is a compound in which an oxygen atom or a nitrogen atom is bonded to each of two adjacent carbon atoms, and since it has at least two asymmetric carbon atoms in one molecule, it exists in multiple optical isomers. The optically active compound (Z) is one of the chemical structures widely used in the fields of pharmaceutical and agricultural chemical development, and can also be used as a ligand for a transition metal catalyst used in an asymmetric reaction.
In the formula, X represents -O- or ^-NRf- , Y represents -O-, -NRg- or ^-S- , ^Ra , ^Rb , ^Rc , ^Rd , ^Re , ^Rf and ^Rg each independently represent a hydrogen atom or an organic group, and an asterisk indicates that the carbon atom is an asymmetric carbon.

Specific examples of compound (Z) include 1,2-diols (when X and Y are both -O-), 1,2-aminoalcohols (when X is -O- and Y is -NH-, or when X is -NH- and Y is -O-), 1,2-diamines (when X and Y are both -NH-), 1,2-mercaptoalcohols (when X is -O- and Y is -S-), and 1,2-mercaptoamines (when X is -NH- and Y is -S-).

To date, many studies have been conducted on methods for producing optically active compound (Z). Among these, the method of obtaining optically active compound (Z) by reacting a readily available compound having an epoxide structure or aziridine structure with a nucleophile and stereoselectively opening the ring is highly atom-efficient and useful.

For example, methods for obtaining optically active compound (Z) by reacting a compound having an epoxide structure with a nucleophile include (A) optical resolution of a racemate (e.g., Patent Documents 1-3, Non-Patent Documents 1 and 2), (B) introducing an asymmetric carbon atom at another position and separating the resulting diastereomers (e.g., Patent Document 4, Non-Patent Document 3), and (C) asymmetric ring-opening reaction of a compound having an epoxide structure with a nucleophile in the presence of an optically active catalyst (e.g., Patent Document 5, Non-Patent Documents 4-6). In particular, known examples of method (A) include a method using an optically active acid as a resolving agent, a separation method using column chromatography with an optically active packing material, and a method using enzymes derived from animals or microorganisms.

In addition, methods for obtaining compound (Z) by reacting a compound having an aziridine structure with a nucleophile include (D) a method of optically resolving a racemate (e.g., Patent Document 6) and (E) a method of performing an asymmetric ring-opening reaction of a compound having an aziridine structure with a nucleophile in the presence of an optically active catalyst (e.g., Non-Patent Documents 7 and 8).

However, methods (A), (B), and (D) do not achieve a theoretical yield exceeding 50%, and require the use of a resolving agent in an amount equal to the product or purification using a large-capacity column, making them unsuitable for industrial production.

Methods (C) and (E), on the other hand, can produce the desired optically active compound (Z) in a short process, but in many cases, a metal catalyst or a strong acid catalyst is used as the optically active catalyst. Therefore, methods (C) and (E) are limited in the compounds or nucleophiles containing a heterocyclic three-membered ring structure that can be used, and require a costly metal catalyst recovery process.

Patent No. 4406483 Patent No. 4406482 U.S. Pat. No. 5,981,267 Japanese Patent Application Publication No. 9-157258 Japanese Patent Application Laid-Open No. 2003-206266 JP 2011-83934 A Patent No. 6630667 Patent No. 6678442

Tetrahedron, 56, 9773-9779 (2000) Synth. Commun. , 29, 1369-1377 (1999) J. Med. Chem. 41, 38-45 (1998) Tetrahedron: Asymmetry, 9, 1747-1752 (1998) Bull. Chem. Soc. Jpn. , 61, 1213-1220 (1988) Chemistry Letters, 36, 34-35 (2007) Org. Biomol. Chem. , 9, 6205-6207 (2011) J. Org. Chem. , 68, 5160-5167 (2003) Biosci. Biotechnol. Biochem. 65(10), 2249-2258 (2001) Bull. Chem. Soc. Jpn. 91, 678-683 (2018) Molecules 25, 3197 (2020) Chem. Eur. J. 22, 11543-11548 (2016) Eur.J.Org.Chem.3849-3869 (2001)

The present inventors have previously found that the asymmetric ring-opening reaction of epoxides or aziridines with nucleophiles proceeds in the presence of plant-processed products (Patent Document 7, Non-Patent Documents 10 and 11). They also found that the catalytic activity of plant-processed products is improved by treating them with enzymes (Patent Document 8).

However, because the active chemical structure of the catalyst is unknown, it has been discovered that obtaining a more highly active catalyst is difficult, and that there is a problem of the likelihood of contamination with impurities derived from processed plant products. Furthermore, processed plant products often have very large molecular weights (for example, the molecular weight of SOYAFIBE S-DN, a water-soluble soybean polysaccharide, is 100,000 to 850,000 (Non-Patent Document 9)), which has led to handling issues such as a tendency for the catalyst to gel when used as a catalyst and for the catalyst to adhere to the container. Furthermore, it has been found that the amount of product recovered decreases when the catalyst gels or adheres to the container. Therefore, an object of the present invention is to provide a new catalyst that can promote the asymmetric ring-opening reaction of epoxides or aziridines with nucleophiles.

The inventors removed lipids and soluble polysaccharides from processed plant products to obtain crude pectic polysaccharides, and then repeatedly treated and purified the crude pectic polysaccharides with enzymes (e.g., lipase, galactanase), acid, alkali, or ion exchange resins. This led to the discovery of the active substance of the catalyst and the completion of this invention.

That is, the present invention provides the following [1] to [30].
[1] A polysaccharide having a degree of polymerization of 16 to 40, consisting of only galactose or consisting of only galactose and 1 to 3 molecules of arabinose, in which the sugar units are linked in an unbranched chain.
[2] A polysaccharide comprising the polysaccharide according to [1] as a partial structure, having a sugar composition containing 90 to 99.5 mol % of galactose and 0.5 to 10 mol % of arabinose, and having an average molecular weight of 2,500 to 9,000.
[3] A polysaccharide comprising the polysaccharide according to [1] as a partial structure, having a sugar composition containing 80 to 98 mol % of galactose and 2 to 20 mol % of arabinose, and having an average molecular weight of 10,000 to 60,000.
[4] A polysaccharide having a degree of polymerization of 16 to 40, which is bound in an unbranched chain, and which is composed only of galactose, or which is composed only of galactose and 1 to 3 molecules of arabinose, and which is an amino sugar in which the terminal galactose is reductively aminated.
[5] A composition containing the polysaccharide according to any one of [1] to [4].
[6] The composition according to [5] for use as a catalyst.
[7] The composition according to [6], for use as a catalyst for promoting the asymmetric ring-opening reaction of a compound having an oxirane, aziridine or thiirane ring with a nucleophile.
[8] Use of a composition containing the polysaccharide according to any one of [1] to [4] as a catalyst.
[9] The use according to [8], wherein the catalyst promotes an asymmetric ring-opening reaction of a compound having an oxirane, aziridine, or thiirane ring with a nucleophile.
[10] A step of mixing a processed plant product with an organic solvent, filtering out a solid material, and obtaining a defatted sample;
mixing the defatted sample with water to remove soluble polysaccharides;
A method for producing crude pectic polysaccharides, comprising the step of heating the sample from which soluble polysaccharides have been removed under acidic conditions, under conditions in which ethylenediaminetetraacetic acid has been added, or under conditions in which ammonium oxalate has been added, to obtain crude pectic polysaccharides.
[11] Use of the crude pectic polysaccharide obtained by the method according to [10] as a catalyst or a raw material for a catalyst.
[12] The use according to [11], wherein the catalyst promotes an asymmetric ring-opening reaction of a compound having an oxirane, aziridine, or thiirane ring with a nucleophile.
[13] A compound represented by formula (1) in the presence of a polysaccharide according to any one of [1] to [4], a crude pectic polysaccharide obtained by the method according to [10], or a galactan having 16 to 24 galactoses linked in a β1,4 bond,
wherein X is —O—, —NR—, or —S—;
R is a hydrogen atom, an optionally substituted C _1-6 alkyl group, an optionally substituted C _3-6 cycloalkyl group, an optionally substituted C 2-6 alkenyl group, an optionally substituted C _3-6 cycloalkenyl group, an optionally substituted C _2-6 alkynyl group, an optionally substituted C _6-10 aryl group, an optionally substituted C _1-6 alkylcarbonyl group, an optionally substituted C _6-10 arylcarbonyl group, an optionally substituted C _1-6 alkylsulfonyl group or _{a C 6-10 arylsulfonyl} group;
R ¹ , R ² , R ³ and R ⁴ are each independently a hydrogen atom, an optionally substituted C _1-6 alkyl group, an optionally substituted C _3-6 cycloalkyl group, an optionally substituted C _2-6 alkenyl group, an optionally substituted C _3-6 cycloalkenyl group, an optionally substituted C _2-6 alkynyl group or an optionally substituted C _6-10 aryl group;
^R2 and ^R3 may be bonded to each other to form a cyclic structure.
reacting a compound represented by formula (2) with
wherein Y is —O—, —NR ⁶ — or —S—;
R ⁵ and R ⁶ are each independently a hydrogen atom, an optionally substituted C _1-6 alkyl group, an optionally substituted C _3-6 cycloalkyl group, an optionally substituted C _2-6 alkenyl group, an optionally substituted C _3-6 cycloalkenyl group, an optionally substituted C _2-6 alkynyl group, or an optionally substituted C _6-10 aryl group;
^R5 and ^R6 may be bonded to each other to form a cyclic structure;
However, the compound represented by formula (2) is not water or hydrogen sulfide.]
A method for producing a compound represented by formula (3), comprising the step of obtaining a compound represented by formula (3).
wherein X, Y, R ¹ , R ² , R ³ , R ⁴ and R ⁵ are as defined above.
[14] The method according to [13], wherein the compound represented by formula (1) is a compound represented by formula (1a):
wherein X is —O—, —NR—, or —S—;
R ¹ and R ⁴ are each independently a hydrogen atom, an optionally substituted C _1-6 alkyl group, an optionally substituted C _3-6 cycloalkyl group, an optionally substituted C _2-6 alkenyl group, an optionally substituted C _3-6 cycloalkenyl group, an optionally substituted C _2-6 alkynyl group, or an optionally substituted C _6-10 aryl group;
R ^2a and R ^3a are each independently a C _1-6 alkylene group, a C _1-6 oxyalkylene group, a C _3-6 cycloalkylene group, a C _2-6 alkenylene group, a C _3-6 cycloalkenylene group, a C _2-6 alkynylene group, or a C _6-10 arylene group.]
[15] The method according to [13] or [14], wherein the compound represented by formula (2) is a compound represented by formula (2a):
[In the formula, R ^5a and R ^6a each represent an optionally substituted C _1-6 alkylene group, an optionally substituted C _1-6 oxyalkylene group, an optionally substituted C _3-6 cycloalkylene group, an optionally substituted C _2-6 alkenylene group, an optionally substituted C _3-6 cycloalkenylene group, an optionally substituted C _2-6 alkynylene group, or a C _6-10 arylene group.]
[16] The method according to any one of [13] to [15], wherein the compound represented by formula (2) has an asymmetric center and is one or a mixture of optical isomers.
[17] In the presence of the polysaccharide according to any one of [1] to [4], a crude pectic polysaccharide obtained by the method according to [10], or a galactan having 16 to 24 galactose units linked in β1,4 bonds, a mixture of at least one compound represented by formula (a) or (b) is prepared.
[wherein R ¹ , R ² , R ³ and R ⁴ each independently represent a hydrogen atom, an optionally substituted C _1-6 alkyl group, an optionally substituted C _3-6 cycloalkyl group, an optionally substituted C _2-6 alkenyl group, an optionally substituted C 3-6 cycloalkenyl group, an optionally substituted C _2-6 alkynyl group or _{an optionally substituted C 6-10 aryl} group;
^R2 and ^R3 may be bonded to each other to form a cyclic structure.
reacting a compound represented by formula (2) with
wherein Y is —O—, —NR ⁶ — or —S—;
R ⁵ and R ⁶ are each independently a hydrogen atom, an optionally substituted C _1-6 alkyl group, an optionally substituted C _3-6 cycloalkyl group, an optionally substituted C _2-6 alkenyl group, an optionally substituted C _3-6 cycloalkenyl group, an optionally substituted C _2-6 alkynyl group, or an optionally substituted C _6-10 aryl group;
^R5 and ^R6 may be bonded to each other to form a cyclic structure;
However, the compound represented by formula (2) is not water or hydrogen sulfide.]
A method for producing a compound represented by formula (3), comprising the step of obtaining a compound represented by formula (3).
wherein Y, R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are as defined above.
[18] (1) a step of reacting 7-oxabicyclo[4.1.0]heptane (also referred to as 1,2-epoxycyclohexane) and cyclopropylamine in the presence of the polysaccharide according to any one of [1] to [4], a crude pectic polysaccharide obtained by the method according to [10], or a galactan having 16 to 24 β1,4-linked galactoses to obtain (1R,2R)-2-cyclopropylamino-1-cyclohexanol; (2a) a step of reacting (1R,2R)-2-cyclopropylamino-1-cyclohexanol with a carbonylation reagent, followed by further reaction with protected or unprotected 6-(3-aminopropoxy)-2(1H)-quinolinone; or (2b) a step of reacting protected or unprotected 6-(3-aminopropoxy)-2(1H)-quinolinone with a carbonylation reagent. and a step of reacting unprotected 6-(3-aminopropoxy)-2(1H)-quinolinone with a carbonylation reagent, followed by further reaction with (1R,2R)-2-cyclopropylamino-1-cyclohexanol, and when protected 6-(3-aminopropoxy)-2(1H)-quinolinone is used, further deprotection is performed to obtain (-)-6-[3-[3-cyclopropyl-3-[(1R,2R)-2-hydroxycyclohexyl]ureido]propoxy]-2(1H)-quinolinone.
[19] A method for producing the polysaccharide according to [3], comprising purifying a processed plant product or a crude pectic polysaccharide obtained by the method according to [10] by treatment with an enzyme or alkali, fractionation by addition of an organic solvent, or a combination of fractionation by addition of an organic solvent and fractionation utilizing differences in solubility due to temperature.
[20] A method for producing the polysaccharide according to [2], comprising fractionating the polysaccharide according to [3] by treating it with an enzyme or acid, or by adding an organic solvent.
[21] A method for producing the polysaccharide according to [2], comprising purifying the processed plant product or the crude pectic polysaccharide obtained by the method according to [10] by treatment with an enzyme, fractionation by addition of an organic solvent, treatment with an anion exchange resin, gel filtration chromatography, or any combination thereof.
[22] A method for producing the polysaccharide according to [1], comprising purifying the polysaccharide according to [2] by a combination of anion exchange column chromatography, gel filtration chromatography, and HPLC.
[23] Galactan consisting of 16 to 24 galactose units linked in β1,4 bonds.
[24] Use of galactan in which 16 to 24 galactoses are β1,4-linked as a catalyst.
[25] The use according to [24], wherein the catalyst promotes an asymmetric ring-opening reaction of a compound having an oxirane, aziridine, or thiirane ring with a nucleophile.
[26] A method for producing a galactan in which 16 to 24 galactoses are β1,4-linked, comprising:
A method comprising the step of reacting a compound represented by formula (14) with a compound represented by formula (16) to obtain a compound represented by formula (17).
[In the formula, R ^{1 A} is an optionally substituted alkylcarbonyl group, R 1 ^B is an optionally substituted aralkyl group, R 1 ^C is an optionally substituted arylcarbonyl group, R 1 ^D is an optionally substituted aryl group, R 1 ^E is an optionally substituted aralkyl group, and n is an integer of 13 to 21.]
[27] The method according to [26], wherein R ^C is a toluoyl group.
[28] The method according to [26] or [27], further comprising a step of hydrolyzing the compound represented by formula (13) to obtain a compound represented by formula (15).
[In the formula, R ^A is an optionally substituted alkylcarbonyl group, R ^B is an optionally substituted aralkyl group, R ^C is an optionally substituted arylcarbonyl group, and R ^F is an optionally substituted aryl group.]
[29] The method according to any one of [26] to [28], further comprising a step of reacting the compound represented by formula (13) with R ^D SH in the presence of a Lewis acid to obtain the compound represented by formula (14).
[In the formula, R ^A is an optionally substituted alkylcarbonyl group, R ^B is an optionally substituted aralkyl group, R ^C is an optionally substituted arylcarbonyl group, R ^D is an optionally substituted aryl group, and R ^F is an optionally substituted aryl group.]
[30] The method according to any one of [26] to [29], further comprising a step of reacting a compound represented by formula (11) with a compound represented by formula (12) in the presence of a Lewis acid to obtain a compound represented by formula (13).
[In the formula, R ^A is an optionally substituted alkylcarbonyl group, R ^B is an optionally substituted aralkyl group, R ^C is an optionally substituted arylcarbonyl group, R ^D is an optionally substituted aryl group, and R ^F is an optionally substituted aryl group.]

The present invention provides a new catalyst that can promote the asymmetric ring-opening reaction of epoxides or aziridines with nucleophiles. Furthermore, by further purifying inexpensively available plant products, the present invention can provide a catalyst with a higher specific activity than the catalysts disclosed in Patent Documents 7-8 and Non-Patent Documents 10-11, which can also be reused. Furthermore, the present invention makes it easier to recover the product and catalyst than in reactions using plant products. Furthermore, it can provide a catalyst with a lower amount of impurities (e.g., allergens) that are problematic in pharmaceutical manufacturing.

1 shows photographs of SDS-PAGE gels of fractions in each purification step of Example A3. 1 shows the amino acid sequence of rhamnogalacturonate lyase from Aspergillus luchuensis mut.kawachii NBRC 4308 in Example A3, where the underlined amino acids are those that overlap with the peptide of LipRGL. 1 is a chromatogram showing the time course of digestion of LP-LMW with Enzyme A in Example A6. Graph (a) shows the catalytic activity of the LP-LMW-derived fraction and the AL-LMW-derived fraction in Example A6(7), and graph (b) shows the stereoselectivity of the LP-LMW-derived fraction and the AL-LMW-derived fraction in Example A6(7). 1A and 1B are graphs showing the catalytic activity and stereoselectivity of each NG fraction in Example A9, respectively; 1 is a graph showing the arabinose content of each NG fraction in Example A9. 1(a) is a chromatogram of the "fraction NG3-4 kDa" obtained by HILIC-HPLC in Example A10, and FIG. 1(b) is a graph showing the chromatograms of each fraction after separation. (a) is a chromatogram of "fraction NG 2-3 kDa" in Example A10(3) when separated by HILIC-HPLC. (b) is a chromatogram of "pool a" and "pool b" in Example A10(3) when purified by preparative HPLC. (c) is a graph showing superimposed chromatograms of HILIC-HPLC analysis of each fraction after separation. 1 is a graph showing the catalytic activity and stereoselectivity of fractions corresponding to the major peak and minor peak in Example A10(5). 1 is a chromatogram of ODS-HPLC fractionation in Example A10(5). The dashed squares indicate fractionated peaks. 1 is a graph showing superimposed chromatograms of ODS-HPLC analysis of each sample after ODS-HPLC fractionation in Example A10(6). 1 is a chromatogram of ODS-HPLC preparative in Example A11. 1 is a graph showing a chromatogram of ODS-HPLC analysis performed before ODS-HPLC fractionation in Example A11 and a chromatogram of ODS-HPLC analysis of each sample obtained by fractionating each of peaks C, D, E, F, G, H, and I, superimposed on each other. Graph (a) shows the catalytic activity of DP16 to DP25 in Example A11, and graph (b) shows the stereoselectivity of DP16 to DP25 in Example A11. 1(a) is a chromatogram showing the results of HPLC analysis (RI detection) of NG-ABEE and the sample (NG) before modification in Example A12. 1(b) is a chromatogram showing the results of HPLC analysis (UV detection) of NG-ABEE and the sample (NG) before modification in Example A12. 1 is a ¹ H-NMR spectrum of AL-LMW in Example B1. 1 is a ¹³ C-NMR spectrum of AL-LMW in Example B1. 1 shows GC spectra obtained by detecting m/z 118 after derivatization of the 19.5-mer and 20-mer in the analysis of sugar chain binding positions in Example B2. 1 is a photograph showing the state of each reaction vessel before the toluene layer is collected in Example C4.

The present invention is described in detail below.

In this specification, galactose is also referred to as "Gal" and arabinose as "Ara."

As used herein, "unbranched chain" means that each sugar unit (galactose, arabinose) except for the terminal sugar unit is bonded to two adjacent sugar molecules.

First Embodiment
The first embodiment of the present invention is a polysaccharide having a degree of polymerization of 16 to 40, consisting of only galactose or consisting of only galactose and 1 to 3 molecules of arabinose, in which the sugar units are linked in an unbranched chain, for example, by β1,4 bonds.

The polysaccharide of this embodiment may be a galactan consisting of 16 to 40 galactose molecules, or an arabinogalactan in which 1 to 3 of the 16 to 40 galactose molecules are replaced with arabinose.

The polysaccharides of this embodiment can be prepared from crude pectic polysaccharides obtained by removing lipids and soluble polysaccharides from processed plant products, or from processed plant products. The polysaccharides of this embodiment can also be obtained by repeatedly treating crude pectic polysaccharides or processed plant products with enzymes (e.g., lipase, rhamnogalacturonate lyase, galactanase), acids, or alkalis; fractionation by adding organic solvents; fractionation utilizing differences in solubility due to cooling; or purification by column chromatography (e.g., anion exchange, gel filtration, HPLC). The polysaccharides of this embodiment may also be artificially synthesized using organic chemistry techniques.

Second Embodiment
A second embodiment of the present invention is a polysaccharide comprising the polysaccharide according to the first embodiment as a partial structure, having a sugar composition comprising 90 to 99.5 mol % of galactose and 0.5 to 10 mol % of arabinose, and having an average molecular weight of 2500 to 9000.

The polysaccharide according to this embodiment may have a sugar composition containing 90 to 99.5 mol % galactose and 0.5 to 10 mol % arabinose. It may consist solely of galactose and arabinose, or may further contain other monosaccharides such as rhamnose, fructose, xylose, glucose, and galacturonic acid. Galactose, arabinose, and other monosaccharides may each independently be either furanose or pyranose.

The polysaccharide of this embodiment has an average molecular weight of 2500 to 9000, preferably 2700 to 8000, and more preferably 2900 to 7000.

Third Embodiment
A third embodiment of the present invention is a polysaccharide comprising the polysaccharide of the first embodiment as a partial structure, having a sugar composition comprising 80 to 98 mol % of galactose and 2 to 20 mol % of arabinose, and having an average molecular weight of 10,000 to 60,000.

The polysaccharide according to this embodiment may have a sugar composition containing 80-98 mol% galactose and 2-20 mol% arabinose, and may be composed solely of galactose and arabinose, or may further contain other monosaccharides such as rhamnose, fructose, xylose, glucose, and galacturonic acid. Galactose, arabinose, and other monosaccharides may each independently be either furanose or pyranose.

The polysaccharide of this embodiment has an average molecular weight of 10,000 to 60,000, preferably 11,000 to 55,000.

The polysaccharides according to the first to third embodiments can be produced as follows. For more specific details, please refer to the Examples of the present application. In the procedures described below, the method of removing insoluble matter or obtaining precipitates by centrifugation may be replaced by decantation or filtration (natural filtration, centrifugal filtration, pressure filtration, or vacuum filtration). Furthermore, the method of purifying using an anion exchange resin column in the method described below may be performed by adding anion exchange resin to the pre-purification sample in a batchwise manner, followed by filtration.

(1) Defatting Step The processed plant product is mixed with an organic solvent and the solid matter is filtered off repeatedly to remove fat-soluble components from the processed plant product and obtain a defatted sample. To more efficiently remove fat-soluble components, the processed plant product is preferably pulverized into powder form. Examples of organic solvents include halogenated hydrocarbons such as dichloromethane and chloroform, alcohols such as methanol and ethanol, and mixtures of these. The temperature in the defatting step may be room temperature (e.g., 0°C to 30°C).

<Plant processed products>
In this specification, the processed plant product refers to a powder or extract obtained by processing a part of a plant.

In the case of edible plants, the term "plant" refers to plants (including algae) generally known as plants whose parts can be eaten by humans. Examples of edible plants include plants classified as grains, legumes, vegetables, fruits, or potatoes, and parts of edible plants can be appropriately selected from the whole fruit, pulp, pericarp, stem (stalk-like portion in the case of algae), seeds, germ, root (appressorium in the case of algae), bulbs, and leaves. In addition, the remainder (inedible portion) after harvesting the edible portion can also be considered part of an edible plant. Specific examples of edible plants include those from the family Magnoliaceae (e.g., star anise), family Malvaceae (e.g., okra, baobab), family Carapaceae (e.g., papaya), family Brassicaceae (e.g., broccoli, Japanese mustard spinach, radish), family Moringa (e.g., moringa), family Cucurbitaceae (e.g., pumpkin), family Asteraceae (e.g., Jerusalem artichoke, burdock, stevia, mugwort), family Lauraceae (e.g., Kuromoji, bay leaf, Ceylon cinnamon), and family Houttuynia cordata (e.g., Houttuynia cordata). Mi), Lamiaceae (e.g., perilla), Oleaceae (e.g., olive), Apiaceae (e.g., angelica, carrot), Ebacaceae (e.g., persimmon), Camellia (e.g., tea plant), Convolvulaceae (e.g., sweet potato, purple sweet potato), Solanaceae (e.g., tobacco, tomato, potato), Amaranthaceae (sugar beet, spinach), Rosaceae (e.g., almond, apple), Urticaceae (e.g., nettle leaf), Moraceae (e.g., mulberry, fig), Myrtaceae Family (e.g., clove), Fagaceae (e.g., Quercus salicina), Juglandaceae (e.g., walnut), Fabaceae (e.g., soybean, black bean, red kidney bean, pea, Cassia japonica, Jack bean), Meliaceae (e.g., neem), Anacardiaceae (mango, pistachio, cashew nut), Ilex family (e.g., yelpa mate), Rubiaceae (e.g., gardenia), Poaceae (e.g., wheat), Xanthomonas arborescens (e.g., Aloe arborescens), Lycoris radiata Examples of suitable plants include plants from the following families: Zingiberaceae (e.g., onion, garlic, leek), Arecaceae (e.g., turmeric), Palmaceae (e.g., coconut), Cupressaceae (e.g., juniper berry), Equisetaceae (e.g., horsetail), Rutaceae (e.g., mandarin orange, yuzu, flower yuzu, pomelo), Nelumbaceae (e.g., lotus root), Actinidiaceae (e.g., kiwi), Sargassumaceae (e.g., hijiki), Strigataceae (e.g., laver), and Funoriaceae (e.g., funori). Edible plants are preferably selected from the group consisting of: Melastomataceae, Papayaaceae, Moringaceae, Cucurbitaceae, Asteraceae, Houttuyniaceae, Lamiaceae, Apiaceae, Theaceae, Myrtaceae, Fagaceae, Fabaceae, Amaryllidaceae, Palmaceae, and Equisetaceae. Note that leeks are sometimes classified as members of the Alliaceae family. The "plant" may also be non-edible, such as tobacco.

The term "processing" as used above refers to processes such as drying, heating, roasting, frying, fermenting, or removing unnecessary parts, as needed, followed by grinding into powder or extracting the components. The processed plant products also include powders obtained by extracting the essence of edible plants and then grinding the dried product. Therefore, the tea may be green tea or black tea. The soybeans may be soybean flour or natto.

The processed plant products may be commercially available in powder or liquid form, or may be commercially available processed products that are crushed into powder as appropriate. Commercially available processed plant products include soybean flour, defatted soybean flour (e.g., Fujipro F (trade name, manufactured by Fuji Oil Co., Ltd.), Sunrich F (trade name, manufactured by Showa Sangyo Co., Ltd.), Soya Flour FT-N (trade name, manufactured by Nisshin Oillio Co., Ltd.), S-Sun Meat Tokushu (trade name, manufactured by Ajinomoto Co., Inc.), Honen Soy Pro (trade name, manufactured by J-Oil Mills Co., Ltd.), water-soluble soybean polysaccharides (e.g., Soya Five S-DN (trade name, manufactured by Fuji Oil Co., Ltd.)), and processed soybean products such as okara (soybean pulp). Soybean flour, Soya Flour FT-N, Soya Five S-DN, or okara are more preferred.

(2) Removal of soluble polysaccharides The defatted sample is mixed with water, centrifuged, and the water is removed to obtain a sample from which soluble polysaccharides have been removed. Tap water, purified water, or RO water (water filtered through a reverse osmosis membrane) can be used as the water. The temperature in this step may be from room temperature to a heated state (e.g., 0°C to 50°C). The centrifugal force used in centrifugation may be, for example, 3,000 to 20,000 × g.

(3) Preparation of Crude Pectic Polysaccharides Crude pectic polysaccharides are obtained from the sample from which soluble polysaccharides have been removed by the acetic acid method, hydrochloric acid method, EDTA (N,N,N',N'-ethylenediaminetetraacetic acid) method, or oxalic acid method. Specifically, the sample from which soluble polysaccharides have been removed is suspended in water, and acetic acid, hydrochloric acid, EDTA, or ammonium oxalate is added. The suspension is then heated to 75-100°C and vigorously stirred for 1-4 hours, or heated in an autoclave at 121°C for 30 minutes to 2 hours. The mixture is then cooled to room temperature and centrifuged. Ethanol is added to the separated precipitate, followed by stirring and centrifuging again. The resulting precipitate is dissolved in water, and the supernatant is collected and lyophilized. The centrifugal force of the centrifugation may be, for example, 3,000-20,000 x g.

The resulting crude pectic polysaccharides can be used as catalysts for asymmetric ring-opening reactions. By converting processed plant products into crude pectic polysaccharides, catalytic activity (specific activity per unit weight) is often improved compared to when processed plant products are used directly as catalysts, broadening the range of plant products that can be used as catalysts. Furthermore, processed plant products from the Poaceae, Cucurbitaceae, Solanaceae, Brassicaceae, and Rosaceae families, which have previously been problematic for practical use due to their low stereoselectivity, have been found to exhibit stereoselectivity of 30% ee or higher. Furthermore, when used as raw materials in the second and third embodiments, higher yields were achieved than when processed plant products were used as raw materials.

(4) Enzyme Treatment of Processed Plant Products A processed plant product or crude pectic polysaccharide produced from a processed plant product is treated with an enzyme (e.g., lipase, rhamnogalacturonate lyase (RGL), galactanase), and insoluble matter is removed by centrifugation. An organic solvent (e.g., acetone) is added to the supernatant, and the mixture is stirred and then centrifuged again. Water is added to the resulting precipitate, and insoluble matter is removed by centrifugation. The resulting supernatant is dialyzed using a dialysis tube with a specified membrane pore size, and the liquid remaining in the tube is freeze-dried. The resulting polysaccharide (LP-LMW) may correspond to the polysaccharide according to the third embodiment.

(5) Alkali Treatment of Processed Plant Products A processed plant product or crude pectic polysaccharide produced from a processed plant product is heated and stirred at 50 to 100°C in the presence of an alkali (e.g., sodium hydroxide, potassium hydroxide), or heated to 100 to 121°C in an autoclave. The resulting reaction solution is neutralized and then centrifuged. An organic solvent (e.g., ethanol, acetone) is added to the resulting supernatant, and the resulting precipitate is collected by centrifugation. Water is added to the resulting precipitate to dissolve it, and the mixture is dialyzed using a dialysis tube with a specified membrane pore size, and the liquid remaining in the tube is freeze-dried. The resulting polysaccharide (AL-LMW) may correspond to the polysaccharide according to the third embodiment.

The alkali may be, for example, an alkali metal hydroxide such as sodium hydroxide or potassium hydroxide. The organic solvent may be, for example, an alcohol such as ethanol, or a ketone such as acetone. The centrifugal force of the centrifugation may be 3,000 to 20,000 x g.

(6) Depolymerization of LP-LMW or AL-LMW (6-1) Depolymerization of Polysaccharides by Galactanase The LP-LMW or AL-LMW obtained in (4) and (5) above is purified using an anion exchange resin column (OH type), and the fraction eluted earlier is collected. The obtained fraction (LMW-D1) may correspond to the polysaccharide according to the third embodiment.

The fraction obtained in (6-1) above is degraded with endo-β1,4-galactanase, followed by dialysis and lyophilization to obtain a low-molecular-weight polysaccharide (LMW-D1deg). The obtained polysaccharide is purified using an anion exchange resin column (OH type), and the fraction that elutes earlier is collected. The obtained polysaccharide (LMW-D1degD1) may correspond to the polysaccharide of the second embodiment.

(6-2) Depolymerization of Polysaccharides with Acid The polysaccharides (LP-LMW or AL-LMW) obtained in (4) and (5) above are hydrolyzed by adding an acid to adjust the pH to 2 to 4, followed by stirring at 70 to 100°C or by heating in an autoclave at 100 to 121°C. Any acid can be used as long as it maintains the pH within the above range, and organic acids (e.g., acetic acid) and mineral acids (e.g., sulfuric acid) can be used. The reaction solution is then neutralized, an organic solvent (e.g., ethanol) is added, and the mixture is stirred. A precipitate is obtained by centrifugation, and the precipitate is purified by dialysis to obtain a polysaccharide (AS-LLMW when sulfuric acid is used, and AA-LLMW when acetic acid is used). The obtained polysaccharides (AS-LLMW and AA-LLMW) may correspond to the polysaccharides according to the second embodiment.

(6-3) Depolymerization of Polysaccharides with Cation Exchange Resins The polysaccharides (LP-LMW or AL-LMW) obtained in (4) and (5) above are dissolved in water, and a cation exchange resin is added (pH is about 2 to 4). The polysaccharide is hydrolyzed by stirring at 70 to 100°C or by heating in an autoclave at 100 to 121°C. Amberlyst may be used as the cation exchange resin. The reaction solution is then neutralized, an organic solvent (e.g., ethanol) is added, and the mixture is stirred. A precipitate is obtained by centrifugation, and the precipitate is purified by dialysis to obtain a polysaccharide (AM-LLMW). The obtained polysaccharide (AM-LLMW) may correspond to the polysaccharide according to the third embodiment.

(6-4) Direct Depolymerization from Processed Plant Products A processed plant product or crude pectic polysaccharides produced from the processed plant product are dissolved in water, heated in an autoclave, and then centrifuged to remove insoluble matter. The resulting supernatant is purified by anion exchange resin column chromatography and dialysis. The resulting fraction (SFD2/3 fraction) is degraded with endo-β1,4-galactanase, fractionated with an organic solvent (e.g., ethanol), dialyzed, and lyophilized to obtain a depolymerized polysaccharide (SFDdegE2 fraction). The resulting polysaccharide is purified by anion exchange resin column chromatography to obtain a depolymerized polysaccharide (SFDdegE2D1 fraction). This is further purified by dialysis and gel filtration column chromatography to obtain a depolymerized polysaccharide (NG fraction). The resulting polysaccharides (SFDdegE2, SFDdegE2D1, and NG fractions) may correspond to the polysaccharides according to the second embodiment.

Fourth Embodiment
The catalyst according to the present invention may be a galactan in which 16 to 24 galactose units are linked by β1,4 bonds. The galactan is a compound represented by formula (18), in which m is 15 to 23. Hereinafter, "a compound represented by formula (18)" and the like will also be referred to as "compound (18)" and the like for convenience.

The galactan according to this embodiment can be produced by a method known in the field of organic chemical synthesis, and may be produced, for example, by deprotecting (i.e., removing R ^A , R ^B , ^{R C} and R ^E ) compound (17) produced by the following method: Compound (17) can be prepared by glycosylation of compound (14) and compound (16).

R ^A is an optionally substituted alkylcarbonyl group, for example, a C _1-6 alkylcarbonyl group optionally substituted with a halogen atom. R ^A is preferably an acetyl group, a chloroacetyl group, a dichloroacetyl group, a trichloroacetyl group, or a bromoacetyl group, more preferably a trichloroacetyl group. ^{R A} is also a protecting group for a hydroxyl group, and can be removed by hydrolysis under acidic or alkaline conditions.

R 1 ^B and R 2 ^E are optionally substituted aralkyl groups, such as a C _1-6 alkyl group substituted with a C _6-10 aryl group. R ^{1 B} is preferably a benzyl group, phenylethyl group, 1-naphthylmethyl group, or 2-naphthylmethyl group, each of which may be substituted with at least one group selected from a C _1-6 alkyl group, a C _1-6 alkoxy group, and a halogen atom. Examples of the C _1-6 alkyl group include a methyl group, an ethyl group, a 1-propyl group, a 2-propyl group, a 1-butyl group, a 2-butyl group, a tert-butyl group, a 1-pentyl group, a 2-pentyl group, a 3-pentyl group, a neopentyl group, a 1-hexyl group, a 2-hexyl group, and a 3-hexyl group. Examples of C _1-6 alkoxy groups include methoxy, ethoxy, 1-propyloxy, 2-propyloxy, 1-butyloxy, 2-butyloxy, tert-butyloxy, 1-pentyloxy, 2-pentyloxy, 3-pentyloxy, neopentyloxy, 1-hexyloxy, 2-hexyloxy, and 3-hexyloxy. Examples of halogen atoms include fluorine, chlorine, bromine, and iodine. R ^{1 B} also serves as a protecting group for a hydroxyl group, and can be removed by subjecting the compound to hydrogenolysis. Hydrogenolysis conditions include, for example, conditions for reacting with hydrogen in the presence of palladium/carbon. The catalyst for the reaction with hydrogen may be any catalyst that can be used in hydrogenolysis.

R ^C is an optionally substituted arylcarbonyl group, such as an optionally substituted C _6-10 arylcarbonyl group. R ^C is preferably a phenylcarbonyl group optionally substituted with at least one selected from a C _1-6 alkyl group, a C _1-6 alkoxy group, and a halogen atom, and more preferably an ortho-, meta-, or para-toluenecarbonyl group (also known as a toluoyl group). When ^{R C} is an optionally substituted arylcarbonyl group, the carbonyl oxygen attacks the anomeric carbon to produce an acyloxonium ion intermediate (also known as neighboring group participation), which can selectively form a β-bond. Furthermore, when synthesizing a polysaccharide to which eight or more monosaccharides are linked, the physical properties of the raw materials and the product are similar, making it difficult to track the progress of the reaction in each step. When R ^C is an optionally substituted arylcarbonyl group, in addition to the selective glycosylation via neighboring group participation described above, it becomes easier to track the progress of the reaction. R 3 ^C is also a protecting group for the hydroxyl group and can be removed by hydrolysis under acidic or alkaline conditions, such as by adding lithium hydroxide to a mixed solvent of tetrahydrofuran and methanol.

R ^D is an optionally substituted aryl group, for example, an optionally substituted C _6-10 aryl. R ^D is preferably a phenyl group optionally substituted with at least one selected from a C _1-6 alkyl group, a C _1-6 alkoxy group, and a halogen atom, and more preferably a 5-tert-butyl-2-methylphenyl group.

n is an integer from 13 to 21, preferably 15 to 19, or 17.

Compound (15) can be obtained by hydrolyzing compound (13). The ester bond represented by R ^A O is cleaved by the hydrolysis. This reaction can be carried out under any conditions that allow hydrolysis of the ester bond represented by R ^A O. For example, acidic or alkaline conditions can be applied, and alkaline conditions are preferred. An example of alkaline conditions is a condition in which 1,4-diazabicyclo[2.2.2]octane (DABCO) is added to a mixed solvent of ethyl acetate and ethanol.
In the formula, R ^A is an optionally substituted alkylcarbonyl group, R ^B is an optionally substituted aralkyl group, R ^C is an optionally substituted arylcarbonyl group, and R ^F is an optionally substituted aryl group.

R ^F is an optionally substituted aryl group, for example, an optionally substituted C _6-10 aryl. R ^F is preferably a phenyl group optionally substituted with at least one selected from a C _1-6 alkyl group, a C _1-6 alkoxy group, and a halogen atom, and more preferably a 4-methoxyphenyl group. R ^F is also a protecting group for a hydroxyl group and can be removed by hydrolysis under acidic or alkaline conditions. Furthermore, when R ^F is a 4-methoxyphenyl group, R ^E can also be removed under oxidative conditions. Examples of oxidative conditions include adding ammonium cerium nitrate (IV) (CAN) to a mixed solvent of acetonitrile, toluene, and water.

Compound (16a) can be obtained by glycosylation of compound (16) and compound (14) to obtain compound (17), followed by conversion of ^ORA to OH. By replacing compound (16) with compound (16a) and repeating these steps, two galactoses can be added at a time to produce a longer galactan structure. That is, compound (18) can be produced by removing the protecting groups ( ^RA , ^RB , ^RC , ^RE ) from compound (17), which is produced by repeating these steps.

In glycosylation reactions using compound (14), an activating agent is added to activate compound (14). The activating agent may be any agent used in glycosylation using thioglycosides, such as methane triflate (MeOTf), N-iodosuccinimide-trimethylsilyl triflate (NIS·TMSOTf), dimethyl(methylthio)sulfonium triflate (DMTST), silver triflate (AgOTf), or an oxidizing agent.

Compound (14) can be obtained by subjecting compound (13) to a glycosylation reaction with R ^D SH in the presence of a Lewis acid.
In the formula, R ^A is an optionally substituted alkylcarbonyl group, R ^B is an optionally substituted aralkyl group, R ^C is an optionally substituted arylcarbonyl group, R ^D is an optionally substituted aryl group, and R ^F is an optionally substituted aryl group.

The Lewis acid can be any acid that can activate the anomeric position, such as boron trifluoride diethyl ether complex.

Compound (13) can be obtained by glycosylation of compound (11) and compound (12) in the presence of a Lewis acid.
In the formula, R ^A is an optionally substituted alkylcarbonyl group, R ^B is an optionally substituted aralkyl group, R ^C is an optionally substituted arylcarbonyl group, R ^D is an optionally substituted aryl group, and R ^F is an optionally substituted aryl group.

In glycosylation reactions using compound (11), an activating agent is added to activate compound (11). The activating agent may be any agent used in glycosylation using a thioglycoside, such as methane triflate (MeOTf), N-iodosuccinimide-trimethylsilyl triflate (NIS·TMSOTf), dimethyl(methylthio)sulfonium triflate (DMTST), silver triflate (AgOTf), or an oxidizing agent.

Fifth Embodiment
In the fifth embodiment of the present invention, a compound represented by formula (1) (hereinafter also referred to as "compound (1)") is reacted with compound (2) in the presence of a polysaccharide according to any one of the first to third and sixth embodiments or a galactan according to the fourth embodiment to obtain compound (3).

<Compound represented by formula (1) (compound (1))>
The compound represented by formula (1) is a compound having an oxirane, aziridine or thiirane structure, and the carbon atom marked with an asterisk (*) in formula (1) may be an asymmetric center.

In formula (1), X is -O-, -NR-, or -S-.

In formula (1), R is a hydrogen atom, an optionally substituted C _1-6 alkyl group, an optionally substituted C _3-6 cycloalkyl group, an optionally substituted C _2-6 alkenyl group, an optionally substituted C _3-6 cycloalkenyl group, an optionally substituted C _2-6 alkynyl group, an optionally substituted C _6-10 aryl group, an optionally substituted C _1-6 alkylcarbonyl group, an optionally substituted C 6-10 arylcarbonyl group, an optionally substituted C _1-6 alkylsulfonyl group or an optionally substituted C _6-10 arylsulfonyl _group .

In formula (1), R ¹ , R ² , R ³ and R ⁴ each independently represent a hydrogen atom, a C _1-6 alkyl group, a C _1-6 alkoxy group, a C _3-6 cycloalkyl group, a C _2-6 alkenyl group, a C _3-6 cycloalkenyl group, a C _2-6 alkynyl group or a C _6-10 aryl group.

The _C1-6 alkyl group means an alkyl group having 1 to 6 carbon atoms. Examples of the _C1-6 alkyl group include a methyl group, an ethyl group, a propan-1-yl group, a propan-2-yl group (isopropyl group), a butan-1-yl group, a butan-2-yl group, a pentan-1-yl group, a pentan-2-yl group, a pentan-3-yl group, a hexan-1-yl group, a hexan-2-yl group, and a 3-hexyl group.

A C _1-6 alkoxy group is a C _1-6 alkyl group bonded to an oxygen atom. Examples of a C _1-6 oxyalkyl group include a methoxy group, an ethoxy group, a propoxy group, a butoxy group, and a pentoxy group.

The C _3-6 cycloalkyl group means a cycloalkyl group having a carbon number of 3 to 6. Examples of the C _3-6 cycloalkyl group include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, and a cyclohexyl group.

A _C2-6 alkenyl group means an alkenyl group having 2 to 6 carbon atoms. Examples of the _C2-6 alkenyl group include a vinyl group, a 1-propen-1-yl group, a 2-propen-1-yl group, a propen-2-yl group, a 2-buten-1-yl group, a 2-buten-2-yl group, a 3-buten-1-yl group, a 2-penten-1-yl group, a 3-penten-1-yl group, a 2-hexen-1-yl group, a 3-hexen-1-yl group, a 4-hexen-1-yl group, and a 5-hexen-1-yl group.

A C _3-6 cycloalkenyl group means a cycloalkenyl group having a carbon number of 3 to 6. Examples of the C _3-6 cycloalkenyl group include a cyclobutenyl group, a cyclopentenyl group, and a cyclohexenyl group.

A C _2-6 alkynyl group means an alkynyl group having a carbon number of 2 to 6. Examples of a C _2-6 alkynyl group include an ethynyl group, a propargyl group, and a 3-butyn-1-yl group.

The C _6-10 aryl group means an aryl group having a carbon number of 6 to 10. Examples of the C _6-10 aryl group include a phenyl group and a naphthyl group.

The _C1-6 alkyl group, _C3-6 cycloalkyl group, _C2-6 alkenyl group, _C3-6 cycloalkenyl group, _C2-6 alkynyl group and _C6-10 aryl group may each be unsubstituted or substituted. Examples of the substituent include a _C1-4 alkyl group, a C2-4 alkenyl group, a _C2-4 alkynyl group, a _C1-4 alkoxy group, an amino group, an imino group, a _nitro group, an oxy group, an oxo group, a nitrile group, a mercapto group or a halogen atom.

Examples of compound (1) include cis-2,3-epoxybutane and 3,4-epoxyhexane.

In formula (1), ^R2 and ^R3 may be bonded to each other to form a cyclic structure. In this case, compound (1) is represented by formula (1a). In formula (1a), X, ^R1 , and ^R4 are defined as in formula (1).

R ^2a and R ^3a are divalent groups represented by replacing hydrogen atoms with single bonds in R ² and R ³ , respectively. That is, R ^2a and R ^3a are each independently a C _1-6 alkylene group, a C _1-6 oxyalkylene group, a C _3-6 cycloalkylene group, a C _2-6 alkenylene group, a C _3-6 cycloalkenylene group, a C _2-6 alkynylene group, or a C _6-10 arylene group.

Examples of groups represented by R ^2a -R ^3a include a 2-oxapropylene group (-CH ₂ OCH ₂ -), a 3-oxopentylene group (-CH ₂ CH ₂ OCH ₂ CH ₂ -), a 3-oxopentylene group (-CH ₂ CH ₂ C(═O)CH ₂ CH ₂ -), and an acetonide group (-CH ₂ -O-C(CH ₃ ) ₂ -O-CH ₂ -).

Specific examples of compounds represented by formula (1a) include 6-oxabicyclo[3.1.0]hexane, 7-oxabicyclo[4.1.0]heptane (also known as 1,2-epoxycyclohexane), 8-oxabicyclo[5.1.0]octane, 3,6-dioxabicyclo[3.1.0]hexane (also known as 3,4-epoxytetrahydrofuran), and 4,4-dimethyl-3,5,8-trioxabicyclo[5.1.0]octane.

<Compound represented by formula (2) (compound (2))>
In formula (2), Y is —O—, —NR ⁶ —, or —S—. That is, compound (2) means an alcohol, an amine, or a thiol. However, water (H ₂ O) and hydrogen sulfide (H ₂ S) are excluded from the scope of compound (2).

In formula (2), ^R5 is a hydrogen atom, an optionally substituted _C1-6 alkyl group, an optionally substituted C3-6 cycloalkyl group, an optionally substituted _C2-6 alkenyl group, an optionally substituted _C3-6 cycloalkenyl group, an optionally substituted _{C2-6 alkynyl} group or an optionally substituted _C6-10 aryl group.

In formula (2), R ⁶ is a hydrogen atom, an optionally substituted C _1-6 alkyl group, an optionally substituted C _3-6 cycloalkyl group, an optionally substituted C _2-6 alkenyl group, an optionally substituted C _3-6 cycloalkenyl group, an optionally substituted C _2-6 alkynyl group or an optionally substituted C _6-10 aryl group.

Examples of _C1-6 alkyl groups include methyl, ethyl, propan-1-yl, propan-2-yl (isopropyl), butan-1-yl, butan-2-yl, pentan-1-yl, pentan-2-yl, pentan-3-yl, hexan-1-yl, hexan-2-yl, and 3-hexyl groups.

A C _1-6 alkoxy group is a C _1-6 alkyl group bonded to an oxygen atom. Examples of a C _1-6 oxyalkyl group include a methoxy group, an ethoxy group, a propoxy group, a butoxy group, and a pentoxy group.

Examples of C _3-6 cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups.

Examples of _C2-6 alkenyl groups include vinyl, 1-propen-1-yl, 2-propen-1-yl, propen-2-yl, 2-buten-1-yl, 2-buten-2-yl, 3-buten-1-yl, 2-penten-1-yl, 3-penten-1-yl, 2-hexen-1-yl, 3-hexen-1-yl, 4-hexen-1-yl, and 5-hexen-1-yl groups.

Examples of C _3-6 cycloalkenyl groups include cyclobutenyl groups, cyclopentenyl groups, and cyclohexenyl groups.

Examples of C _2-6 alkynyl groups include ethynyl groups, propargyl groups, and 3-butyn-1-yl groups.

Examples of C _6-10 aryl groups include phenyl and naphthyl groups.

The _C1-6 alkyl group, _C3-6 cycloalkyl group, _C2-6 alkenyl group, _C3-6 cycloalkenyl group, _C2-6 alkynyl group and _C6-10 aryl group may each be unsubstituted or substituted. Examples of the substituent include a _C1-4 alkyl group, a C2-4 alkenyl group, a _C2-4 alkynyl group, a _C6-10 aryl group, _{a C1-4} alkoxy group, an amino group, an imino group, a nitro group, an oxy group, an oxo group, a nitrile group, a mercapto group or a halogen atom.

In one embodiment, compound (2) has an asymmetric center at ^R5 or ^R6 . When compound (2) has an asymmetric center, compound (2) has optical isomers (enantiomers, diastereomers) derived from the asymmetric center. In this method, a single optical isomer of these optical isomers may be used, or a mixture of any two or more optical isomers (e.g., a racemate) may be used. When compound (2) having an asymmetric center is used, a difference in reaction rate may occur between the optical isomers. Therefore, even if racemic compound (2) is used, one optical isomer may proceed preferentially in this reaction. For example, stereoselectivity can be further enhanced by performing an asymmetric ring-opening reaction using compound (2) having an asymmetric center at ^R5 or ^R6 . Subsequent removal of ^R5 or ^R6 can also produce a product with higher optical purity than when performing an asymmetric ring-opening reaction using compound (2) without an asymmetric center.

Specific examples of compound (2) include methanol, ethanol, 1-propanol, 2-propanol (isopropanol), 1-butanol, 2-butanol, 1-pentanol, 2-pentanol, phenol, ammonia, methylamine, ethylamine, propylamine, 2-propylamine (isopropylamine), 2-pentylamine, 3-pentylamine, cyclopropylamine, cyclobutylamine, cyclopentylamine, cyclohexylamine, tert-butylamine, sec-butylamine, Examples of the methylamine include methylamine (R-, S- and racemic forms), 2-amino-3-methylbutane (R-, S- and racemic forms), 1-cyclohexylethylamine (R-, S- and racemic forms), allylamine, propargylamine, benzylamine, 2-phenylethylamine, aniline, dimethylamine, diethylamine, 4-methoxybenzylamine, 3-methoxypropylamine, 3-ethoxypropylamine, methanethiol, ethanethiol, 1-propanethiol, 2-propanethiol, and butanethiol.

In formula (2), ^R5 and ^R6 may be bonded to each other to form a cyclic structure. In this case, compound (2) is represented by formula (2a). In formula (2a), Y is defined as in formula (2). In formula (2a), ^R5a and ^R6a represent a group formed by bonding to each other.

R ^5a and R ^6a are divalent groups represented by replacing hydrogen atoms with single bonds in R ⁵ and R ⁶ , respectively. That is, R ^5a and R ^6a are each an optionally substituted C _1-6 alkylene group, an optionally substituted C 1-6 oxyalkylene group, an optionally substituted C _3-6 cycloalkylene group, an optionally substituted C _2-6 alkenylene group, an optionally substituted C _3-6 cycloalkenylene group, _{an optionally substituted C 2-6 alkynylene} group, or a C _6-10 arylene group.

Examples of groups represented by R ^5a -R ^6a include a 2-oxapropylene group (-CH ₂ OCH ₂ -), a 3-oxapentylene group (-CH ₂ CH ₂ OCH ₂ CH ₂ -), and a 3-oxopentylene group (-CH ₂ CH ₂ C(=O)CH ₂ CH ₂ -).

Specific examples of compound (2a) include pyrrolidine, piperidine, morpholine, piperazine, homopiperazine, and thiomorpholine.

The _C1-6 alkyl group, _C3-6 cycloalkyl group, _C2-6 alkenyl group, _C3-6 cycloalkenyl group, _C2-6 alkynyl group, and _C6-10 aryl group may each be unsubstituted or substituted. Examples of the substituent include a _C1-4 alkyl group, a _C2-4 alkenyl group, a C2-4 alkynyl group, a _C1-4 alkoxy group, an amino group, an imino group, a _nitro group, a hydroxy group, an oxo group, a nitrile group, a mercapto group, or a halogen atom. Examples of the _C1-4 alkoxy group include a methoxy group, an ethoxy group, a propyloxy group, and a butoxy group.

The amount of compound (2) can be any amount taking into consideration economic efficiency and recoverability. Such an amount is, for example, 0.01 to 100 equivalents, preferably 0.1 to 10 equivalents, and more preferably 0.5 to 2 equivalents, relative to the number of moles of compound (1).

<Compound represented by formula (3) (compound (3))>
Compound (3) is a compound represented by formula (3), in which X, Y, R ¹ , R ² , R ³ , R ⁴ , and R ⁵ are the same as defined above.

Furthermore, compound (3) has a chemical structure corresponding to the structures of compound (1) and compound (2). For example, compound (3) can be represented by the following formula (3a), (3b), or (3c).

Specific examples of compound (3) include 1,2-diols (when X and Y are both -O-), 1,2-aminoalcohols (when X is -O- and Y is -NH-, or when X is -NH- and Y is -O-), 1,2-diamines (when X and Y are both -NH-), 1,2-mercaptoalcohols (when X is -O- and Y is -S-), and 1,2-mercaptoamines (when X is -NH- and Y is -S-).

In the method according to this embodiment, at least one selected from compounds (a), (b), (c), and (d) may be used in place of compound (1). Compounds (a), (b), (c), and (d) may undergo epoxidation in the reaction system to produce compound (1). Compound (1) produced in situ undergoes an asymmetric ring-opening reaction with compound (2) to produce compound (3). Compounds (a) and (c) produce epoxides with the same stereochemistry as each other, and compounds (b) and (d) produce epoxides with the same stereochemistry as each other. The epoxide produced from compound (a) or (c) has the opposite stereochemistry to the epoxide produced from compound (b) or (d).
In the formula, R ¹ , R ² , R ³ , and R ⁴ each independently represent a hydrogen atom, an optionally substituted C _1-6 alkyl group, an optionally substituted C _3-6 cycloalkyl group, an optionally substituted C _2-6 alkenyl group, an optionally substituted C _3-6 cycloalkenyl group, an optionally substituted C _2-6 alkynyl group, or an optionally substituted C _6-10 aryl group, and R ² and R ³ may be bonded to each other to form a cyclic structure. For example, in the presence of the above-mentioned polysaccharide, crude pectic polysaccharide, or galactan, trans-2-chlorohexanol, instead of 7-oxabicyclo[4.1.0]heptane, is mixed with cyclopropylamine to convert trans-2-chlorohexanol to 7-oxabicyclo[4.1.0]heptane, which is then further converted to 2-cyclopropylaminocyclohexanol by a catalytic reaction. For reaction conditions, see the first embodiment.

<Asymmetric ring-opening reaction>
The amount of the catalyst (the polysaccharide according to the first to third and fifth embodiments) can be any amount taking into consideration economic efficiency and recyclability. The amount of such a plant processed product is, for example, 0.01 to 100 times, preferably 0.1 to 10 times, and more preferably 1 to 5 times, the mass of the compound represented by formula (1).

The asymmetric ring-opening reaction according to an embodiment of the present invention may be carried out in a solvent. When carrying out the reaction in a solvent, organic solvents well known in organic synthetic chemistry, such as water, can be used as long as they do not react with compound (1) and compound (2). Examples of such organic solvents include aromatic hydrocarbons such as benzene, toluene, and xylene; hydrocarbons such as hexane, cyclohexane, and heptane; ethers such as diisopropyl ether, tetrahydrofuran, methyl tert-butyl ether, ethyl tert-butyl ether, and cyclopentyl methyl ether; esters such as ethyl acetate and butyl acetate; and halogenated hydrocarbons such as dichloromethane and chloroform. These solvents may be used alone or in combination. When using a combination of two or more solvents, it is preferable to use a mixture of an organic solvent and water. From the standpoints of recoverability, safety, and economy, it is particularly preferable to use a mixture of toluene or heptane with water.

The amount of solvent that can be used in the asymmetric ring-opening reaction, whether a single solvent or a mixed solvent, can be determined in an amount that takes economic efficiency into consideration. The amount of such solvent is, for example, 0 to 100 times, preferably 0.5 to 50 times, and more preferably 2 to 10 times the mass of compound (1) by volume.

The amount of water that can be used in the asymmetric ring-opening reaction can be in the range of 0.05 to 1 times the amount of water relative to the catalyst by mass, and more preferably in the range of 0.20 to 0.50 times the amount of water relative to the catalyst. This range further improves the conversion rate of the reaction and the optical purity of the product.

The reaction temperature is preferably -20°C to 100°C, and from the viewpoint of improving catalytic activity, 30°C to 70°C or 30°C to 50°C is particularly preferred. After the reaction is complete, the catalyst can be filtered off to obtain the corresponding compound (3). The catalyst recovered by filtration can be reused when performing the asymmetric ring-opening reaction of the present invention.

The reaction can be carried out for a time that allows for any desired conversion rate, taking economical efficiency into consideration. Such a reaction time is, for example, 1 to 500 hours, preferably 1 to 100 hours, and more preferably 1 to 48 hours.

After the reaction is complete, the catalyst can be filtered off to obtain compound (3). The resulting compound (3) can be easily purified by standard methods such as crystallization and distillation.

The solvents that can be used for crystallization are not particularly limited as long as they are solvents typically used in organic synthetic chemistry, and examples include hydrocarbons such as hexane, heptane, and cyclohexane; aromatic hydrocarbons such as toluene, benzene, and xylene; and ethers such as diisopropyl ether, methyl tert-butyl ether, ethyl tert-butyl ether, and cyclopentyl methyl ether. These solvents may be used alone or in combination of two or more.

The amount of the above solvent, whether a single solvent or a mixed solvent, can be determined taking economical efficiency into consideration, and is 0.1 to 100 times, preferably 0.5 to 50 times, and more preferably 1 to 10 times the mass of compound (3) by volume.

<Salt formation reaction>
The optical purity of compound (3) obtained by the present invention can be increased by forming a salt with an inorganic or organic acid commonly used in organic synthetic chemistry. Examples of the acid include inorganic acids such as hydrochloric acid, sulfuric acid, sulfurous acid, nitric acid, perchloric acid, chloric acid, iodic acid, and phosphoric acid; and organic acids such as formic acid, acetic acid, lactic acid, oxalic acid, citric acid, maleic acid, fumaric acid, benzoic acid, phthalic acid, salicylic acid, methanesulfonic acid, and toluenesulfonic acid.

To increase the optical purity of compound (3), a salt with an optically active acid may be formed. Examples of such optically active acids include tartaric acid, malic acid, mandelic acid, and phenylglycine, and the acids may be substituted.

When forming the salt, solvents commonly used in organic synthetic chemistry can be used, taking into consideration factors such as economy and recoverability. Examples of such solvents include aromatic hydrocarbons such as benzene, toluene, and xylene; hydrocarbons such as hexane, cyclohexane, and heptane; ethers such as diisopropyl ether, tetrahydrofuran, methyl tert-butyl ether, ethyl tert-butyl ether, and cyclopentyl methyl ether; esters such as ethyl acetate and butyl acetate; halogenated hydrocarbons such as dichloromethane and chloroform; alcohols such as methanol, ethanol, and isopropanol; and water. These solvents may be used alone or in combination.

The amount of solvent used to form the salt can be determined economically, whether it is a single solvent or a mixed solvent. The amount of the solvent is, for example, 0 to 100 times, preferably 0.5 to 50 times, and more preferably 2 to 10 times the mass of compound (1) by volume.

In order to obtain compound (3) of higher purity in the salt formation reaction, it can be purified by methods well known to those skilled in the art.

<Application to drug candidate compounds>
(1R,2R)-2-Cyclopropylamino-1-cyclohexanol (hereinafter also referred to as "Compound A") obtained in the present invention can be reacted with 6-(3-aminopropoxy)-2(1H)-quinolinone (hereinafter also referred to as "Compound B") to produce (-)-6-[3-[3-cyclopropyl-3-[(1R,2R)-2-hydroxycyclohexyl]ureido]propoxy]-2(1H)-quinolinone.

(-)-6-[3-[3-cyclopropyl-3-[(1R,2R)-2-hydroxycyclohexyl]ureido]propoxy]-2(1H)-quinolinone is a substance that possesses two effects in vivo: a strong antithrombotic effect and an inhibitory effect on vascular endothelial hyperplasia. It also has platelet aggregation inhibitory effects, platelet clump dissociation effects, and increased cerebral and peripheral vascularity. Therefore, (-)-6-[3-[3-cyclopropyl-3-[(1R,2R)-2-hydroxycyclohexyl]ureido]propoxy]-2(1H)-quinolinone is useful for the treatment and prevention of thrombotic diseases and arteriosclerotic diseases.

A known method can be used to produce (-)-6-[3-[3-cyclopropyl-3-[(1R,2R)-2-hydroxycyclohexyl]ureido]-propoxy]-2(1H)-quinolinone from Compound A and Compound B. An example of a known method is the method described in Patent Document 4.

When reacting compound A and compound B, as shown in the following formula (Eq. 1), compound A and a carbonylation reagent can be reacted in advance, and then compound B can be reacted. After reacting compound A and a carbonylation reagent, the reaction product may be purified.
[wherein Z represents the residue of a carbonylation reagent.]

Step (i) can be carried out solventlessly or in a solvent. Examples of solvents that can be used in step (i) include ethers such as dioxane, tetrahydrofuran, and diethyl ether; aromatic hydrocarbons such as benzene, toluene, and xylene; halogenated hydrocarbons such as dichloromethane and chloroform; alcohols such as methanol, ethanol, and isopropanol; and polar solvents such as N,N-dimethylformamide, acetone, dimethyl sulfoxide, acetonitrile, and water. These solvents may be used alone or in combination.

Examples of the carbonylation reagent include chloroformates such as phenyl chloroformate, carbonates such as diethyl carbonate, carbonyldiimidazole, phosgene, and triphosgene.

Step (i) is typically carried out at -20 to 150°C, preferably -20 to 100°C.

Step (i) can be carried out in the presence or absence of a basic compound. Examples of basic compounds that can be used in step (i) include inorganic bases such as potassium carbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, sodium bicarbonate, and sodium hydride; and organic bases such as triethylamine, N,N-diisopropylethylamine, imidazole, and pyridine.

In step (i), an additive may be used to promote the reaction more efficiently. Examples of such additives include potassium iodide, sodium iodide, imidazole, 4-dimethylaminopyridine, and 4-pyrrolidinopyridine.

Step (ii) can be carried out without a solvent or in a solvent. The solvents listed in step (i) can be used in step (ii).

Step (ii) is typically carried out at -20 to 150°C, preferably -20 to 100°C.

Step (ii) can be carried out in the presence or absence of a basic compound. The basic compounds that can be used in step (ii) include the basic compounds listed in step (i).

Alternatively, as shown in the following formula (Eq. 2), compound B may be reacted with a carbonylating reagent in advance, and then compound A may be reacted. In this case, each step can be carried out in the same manner as in formula (Eq. 1).
[wherein Z represents the residue of a carbonylation reagent.]

Compound B used in the present invention may be a protected compound B. Protected compound B can be one in which the 1-position of the quinolinone is substituted with a protecting group. Examples of such protecting groups include alkyl groups such as a methoxymethyl group and a benzyl group; substituted silyl groups such as a triethylsilyl group and a triphenylsilyl group; substituted acyl groups such as an acetyl group and a trifluoroacetyl group; and alkoxycarbonyl groups such as a tert-butoxycarbonyl group.

Also, 6-(3-aminopropoxy)-quinoline substituted at the 2-position can be used as protected compound B. Examples of substituents on 6-(3-aminopropoxy)-quinoline substituted at the 2-position include halogen atoms such as fluorine, chlorine, and bromine; alkoxy groups such as methoxy and methoxymethoxy; arylalkyloxy groups such as benzyloxy; acyloxy groups such as acetoxy and pivaloyloxy; and silyloxy groups such as triethylsilyloxy.

When a protected compound B is used in the reaction, it can be deprotected by a known method after the reaction and converted to (-)-6-[3-[3-cyclopropyl-3-[(1R,2R)-2-hydroxycyclohexyl]ureido]-propoxy]-2(1H)-quinolinone. The deprotection conditions used are those described in *Protective Groups in Organic Synthesis*, John Wiley and Sons (1980), and include, for example, acidic conditions, alkaline conditions, and hydrogenation.

Sixth Embodiment
A sixth embodiment of the present invention is a polysaccharide having a degree of polymerization of 16 to 40, which is bound in an unbranched chain, and which is an amino sugar formed by reductively amminating the reducing end galactose of a polysaccharide composed only of galactose or a polysaccharide composed only of galactose and 1 to 3 molecules of arabinose.

The polysaccharide according to this embodiment is an aminopolysaccharide obtained by reductive amination of the terminal galactose in the polysaccharide according to the first embodiment. That is, the aminosugar according to this embodiment has the following partial structure:

In the above formula, R ^X is an organic group, for example, R ^X is an optionally substituted C _1-6 alkyl group, an optionally substituted C _3-6 cycloalkyl group, an optionally substituted C _2-6 alkenyl group, an optionally substituted C 3-6 cycloalkenyl group, an optionally substituted C _2-6 alkynyl group, an optionally substituted C _6-10 aryl group, an optionally substituted C _1-6 alkylcarbonyl group, an optionally substituted C _6-10 arylcarbonyl group, an optionally substituted C _1-6 alkylsulfonyl group or an optionally substituted C _6-10 arylsulfonyl group _. For the definition of each group, see the above description.

R ^X is preferably a p-alkoxycarbonylphenyl group, more preferably a p-ethoxycarbonylphenyl group or a tert-butoxycarbonylphenyl group. When the amino sugar according to this embodiment is produced by reductive amination from the polysaccharide according to the first embodiment, R ^X is derived from the chemical structure of the amine (R ^X -NH ₂ ) used in the reductive amination reaction. For example, when R ^X is a p-ethoxycarbonylphenyl group or a tert-butoxycarbonylphenyl group, the amine used is ethyl p-aminobenzoate (ABEE) or tert-butyl p-aminobenzoate (ABtB), respectively.

Like the polysaccharides of the first to third embodiments, the amino sugars of this embodiment also exhibit catalytic activity and stereoselectivity in asymmetric ring-opening reactions with nucleophiles. Furthermore, the amino sugars of the fifth embodiment are ABEE- or ABtB-conjugated, and therefore can also be used as labels.

The analytical methods used in the following examples and comparative examples are described below.

(1) Catalytic activity measurement method I (100 mg catalyst scale)
A measurement sample (200 mg) was weighed into a flat-bottomed, screw-cap test tube with an inner diameter of 15 mm, and a toluene solution (1.0 mL) of substrate (1.0 M 1,2-epoxycyclohexane, 1.5 M cyclopropylamine) was added. Saturated saline (60 μL) was then added, and the reaction was carried out at 37±1°C with a stir bar and vigorously stirred. Every few hours, 20 μL of the reaction solution was sampled, diluted 5-10 times with toluene, dehydrated with anhydrous magnesium sulfate or anhydrous sodium sulfate, and subjected to GC analysis.

GC analysis was carried out under the following conditions:
Equipment: GC-2014 (Shimadzu Corporation)
Column: BETA DEXTM325 (30 m x 0.25 mm x 0.25 μm)
Flow rate: 94 kPa (inlet pressure)
Split ratio: 1:30
Carrier gas: Helium Column temperature: 120°C
Vaporization chamber temperature: 230°C
Detector: FID (temperature 300°C)

The conversion rate of 1,2-epoxycyclohexane was calculated from the chromatogram areas of the substrate (1,2-epoxycyclohexane) and product (2-cyclopropylamino-1-cyclohexanol) using the effective carbon number method, correcting the area values. The catalytic activity required to produce 1 μmol of 2-cyclopropylamino-1-cyclohexanol from 1,2-epoxycyclohexane per minute was defined as 1 U, and the catalytic activity was expressed as specific activity per mg of catalyst (U/mg).

The stereoselectivity of the catalyst was expressed as the optical purity (%ee) of the product and calculated as follows:

Stereoselectivity (% ee) = {(1R,2R)-2-cyclopropylamino-1-cyclohexanol area - (1S,2S)-2-cyclopropylamino-1-cyclohexanol area} / {(1R,2R)-2-cyclopropylamino-1-cyclohexanol area + (1S,2S)-2-cyclopropylamino-1-cyclohexanol area} x 100

(2) Catalytic activity measurement method II (when measuring only the conversion rate)
5 mg of a polysaccharide sample was weighed into a 2 mL glass container, and 200 μL of a toluene solution of the substrate (0.4 M 1,2-epoxycyclohexane, 0.8 M cyclopropylamine) and 1 μL of saturated saline were added, followed by a reaction at 37°C and 1,000 rpm for 20 hours. After the reaction, the substrate conversion rate (%) and stereoselectivity (%ee) were measured by GC analysis as described in "Catalytic Activity Measurement Method I."

(3) Catalytic activity measurement method III (5 mg catalyst amount)
5 mg of a polysaccharide sample was weighed into a 2 mL glass container, and 100 μL of a toluene solution of the substrate (1.0 M 1,2-epoxycyclohexane, 1.5 M cyclopropylamine) and 6 μL of saturated saline were added. The reaction was carried out at 37°C and 1,000 rpm using a stirrer bar. Every few hours, the catalytic activity (mU/mg) and stereoselectivity (%ee) were measured by GC analysis as described in "Catalytic Activity Measurement Method I."

(4) Catalytic activity measurement method IV (0.1 mg catalyst amount)
0.1 mg of a polysaccharide sample was weighed into a 2 mL glass container, and 100 μL of a substrate toluene solution (1.0 M 1,2-epoxycyclohexane, 1.5 M cyclopropylamine) and 0.2 μL of saturated saline were added, followed by a reaction using a stir bar at 37°C and 1,000 rpm for 20 hours. After the reaction, the catalytic activity (mU/mg) and stereoselectivity (% ee) were measured by GC analysis as described in "Catalytic Activity Measurement Method I."

(5) Catalytic activity measurement method V (5 to 10 mg catalyst amount)
5-10 mg of a polysaccharide sample was weighed into a 2 mL glass container, and 100 μL of a toluene solution of the substrate (1.0 M 4,4-dimethyl-3,5,8-trioxabicyclo[5.1.0]octane, 1.5 M benzylamine) and 6 μL of saturated saline were added. The mixture was stirred at 1,000 rpm using a stirrer bar and reacted at 37° C. Catalytic activity (mU/mg) and stereoselectivity (% ee) were measured every few hours by GC analysis (condition A in Table 45) and HPLC analysis (condition α in Table 44).

(5) Gel filtration HPLC analysis method I
After removing impurities by centrifugation, 100 μL of the 10 mg/mL polysaccharide sample solution was transferred to an HPLC vial and used for measurement. For samples of 10 kDa or greater, a TOSOH TSKgel G3000PW (7.5 mm I.D. x 30 cm, Tosoh Corporation) was used, and for samples of 10 kDa or less, a TOSOH TSKgel G2500PWXL (7.8 mm I.D. x 30 cm, Tosoh Corporation) was used. Measurements were performed using 50 mM sodium acetate buffer (pH 5.0) as the mobile phase, with a sample injection volume of 10 μL, a flow rate of 0.6 mL/min, a column temperature of 35°C, a measurement time of 40 minutes, and an RID detector.

(6) Gel filtration HPLC analysis method II
After removing impurities by centrifugation, 100 μL of the 10 mg/mL polysaccharide sample solution was transferred to an HPLC vial and used for the measurement. The following column was used, and the analytical conditions were: sample injection volume 10 μL, flow rate 0.6 mL/min, column temperature 40°C, measurement time 40 minutes, and RID detector. For samples of 10 kDa or greater, a column consisting of a TOSOH TSKgel G3000PWXL, 7.8 mm I.D. x 30 cm, manufactured by Tosoh Corporation) and a TOSOH TSKgel G4000PWXL, 7.8 mm I.D. x 30 cm, manufactured by Tosoh Corporation was used. A calibration curve was prepared using pullulan (6.3-642 kDa, Shodex Standard P-82, manufactured by Showa Denko K.K.). When measuring samples of 10 kDa or less, a column connected to two columns (TOSOH TSKgel G2500PWXL and TOSOH TSKgel G3000PW, 7.8 mm I.D. x 30 cm, both manufactured by Tosoh Corporation) was used, and PEG 2000-10000 was used to create the calibration curve. The molecular weight calculated from the retention time of the apex of each peak in the chromatogram was used as the average molecular weight.

(7) Monosaccharide Composition Analysis Method: 100 μL of a 1 mg/mL polysaccharide sample solution and 100 μL of 4 M trifluoroacetic acid (TFA, final concentration 2 M) were mixed in a 2 mL screw-cap tube and incubated on a heat block at 120°C for 2 hours. After cooling to room temperature, the solvent and TFA were removed by lyophilization. The resulting (monosaccharified) lyophilized product was dissolved again in 50 μL of purified water to obtain a monosaccharified sample. 165 mg of 4-ethylaminobenzoate (ABEE), 35 mg of sodium cyanoborohydride, 350 μL of methanol, and 41 μL of acetic acid were mixed and heated at 60°C to prepare an ABEE solution. 40 μL of this solution was mixed with 10 μL of the monosaccharified sample solution in a 1.5 mL tube and incubated on a heat block at 80°C for 1 hour. After cooling to room temperature, 200 μL of purified water and 200 μL of chloroform were added to extract impurities into the organic layer, and the aqueous layer was analyzed by HPLC using a TSKGel ODS120H (manufactured by Tosoh Corporation) column and a 50 mM borate buffer (pH 9.0)/acetonitrile mixed solvent (volume ratio 91:9) as the mobile phase at a flow rate of 0.8 mL/min, a column temperature of 35°C, and a UV detection wavelength of 310 nm.

(8) MALDI-TOFMS analysis: The instrument used was a JMS-S3000 Spiral TOF-plus (manufactured by JEOL) and 2,5-dihydroxybenzoic acid was used as the matrix. Measurements were performed in positive ion mode, laser intensity 60, and delay time 350. Spiral mode was used for molecular weights of 3000 or less, and linear mode for molecular weights exceeding 3000.

Example A1: Preparation of crude pectic polysaccharides from plant matter (1) Preparation of defatted sample Approximately 50 g of plant powder was weighed into a 500 mL Erlenmeyer flask, and 300 mL of a chloroform/methanol mixture (volume ratio 9:1) was added. The flask was covered with aluminum foil and vigorously stirred for 1 hour using a stirrer. After suction filtration using a 110 mm diameter suction funnel and a 1000 mL suction bottle, the flask and filtered plant matter were washed with the mixture. The recovered plant matter and 300 mL of the mixture were added to the flask, and after stirring again for 1 hour using a stirrer, the plant matter was recovered by filtration. This process was repeated two more times (total of three times) to obtain a defatted sample.

(2) Preparation of Soluble Polysaccharide-Removed Sample Approximately 30 g of the defatted sample was weighed into a 500 mL Erlenmeyer flask, 300 mL of RO water (water filtered through a reverse osmosis membrane) was added, the flask was covered with aluminum foil, and the mixture was vigorously stirred overnight at 4°C using a stirrer. The Erlenmeyer flask was then heated in a 50°C water bath and vigorously stirred for an additional hour using a stirrer. The mixture was transferred to a 500 mL centrifuge bottle and centrifuged (17,800 × g, 10 minutes, 4°C) to obtain a sample from which soluble polysaccharides had been removed.

(3) Preparation of crude pectic polysaccharides using dilute acetic acid (acetic acid method)
30 g of the soluble polysaccharide-removed sample and 300 mL of 10 mM acetic acid aqueous solution were added to a 500 mL Erlenmeyer flask, covered with aluminum foil, and heated in an autoclave (121°C, 3 hours). The mixture was then transferred to a 500 mL centrifuge bottle and centrifuged (17,800 x g, 10 minutes, 4°C). The resulting supernatant was placed in a 1 L beaker, and ethanol (twice the volume of the supernatant) was added and vigorously stirred using a stirrer. The precipitate was collected by centrifugation (17,800 x g, 10 minutes, 4°C), dissolved in RO water, and the supernatant after centrifugation (17,800 x g, 10 minutes, 4°C) was collected and lyophilized to obtain a crude pectic polysaccharide sample.

(4) Preparation of crude pectic polysaccharides using hydrochloric acid (hydrochloric acid method)
30 g of the soluble polysaccharide-removed sample and 500 mL of RO water were placed in a 1000 mL Erlenmeyer flask, and dilute hydrochloric acid was added to adjust the pH to 1.5. The Erlenmeyer flask was covered with aluminum foil and heated in a 90°C water bath, followed by vigorous stirring for 4 hours using a stirrer. The sample was transferred to a 500 mL centrifuge bottle and centrifuged (17,800 x g, 10 minutes, 4°C). The resulting supernatant was then subjected to ethanol precipitation and lyophilization in the same manner as in (3) above.

(5) Preparation of crude pectic polysaccharides using ethylenediaminetetraacetic acid (EDTA method)
30 g of the soluble polysaccharide-removed sample, 500 mL of 0.01 N hydrochloric acid, and 25 mg of disodium EDTA were sequentially placed in a 1000 mL Erlenmeyer flask. The Erlenmeyer flask was covered with aluminum foil and heated in a 90°C water bath, followed by vigorous stirring using a stirrer for 1 hour. Centrifugation, ethanol precipitation, and freeze-drying were carried out in the same manner as in (3) above.

(6) Preparation of crude pectic polysaccharides using ammonium oxalate (oxalic acid method)
30 g of the soluble polysaccharide-removed sample, 1000 mL of RO water, and 250 mg of ammonium oxalate monohydrate were added to a 2000 mL Erlenmeyer flask, and the pH was adjusted to 3.5 with dilute acetic acid. The Erlenmeyer flask was covered with aluminum foil and heated in a 75°C water bath, followed by vigorous stirring for 1 hour using a stirrer. Centrifugation, ethanol precipitation, and freeze-drying were carried out in the same manner as in (3) above.

The catalytic activity and stereoselectivity of the crude pectic polysaccharides from each plant were measured using "Catalytic Activity Measurement Method I," and the results, along with the yield, are shown in Tables 1 and 2. In the tables, "A" indicates that a defatted sample was used, and "B" indicates that crude pectic polysaccharides prepared by the acetic acid method were used.

Abbreviations for sources in the table: GA: Gaban Co., Ltd., AG: Agristyle Co., Ltd., OR: Oritama Co., Ltd., LE: Leaf Co., Ltd., MK: Mikasa Sangyo Co., Ltd., HC: Healthy Company Co., Ltd., HN: Hinata Co., Ltd., YN: Yamanenen Co., Ltd., OT: Otsuya Shoten Co., Ltd., MI: MI International Co., Ltd., RT: RT Japan Co., Ltd., KY: Healthy Wild Herb Tea Center Co., Ltd., CH: Bikuni Bussan Co., Ltd., JG : Japan Gate Co., Ltd., PA: P@Life Co., Ltd., CB: China Trading Co., Ltd., NT: Mitsui Norin Co., Ltd., OI: Oigawa Tea Garden Co., Ltd., YT: Yamatakeen Co., Ltd., OK: Otsuka Pharmaceutical Co., Ltd., NG: Nippon Garlic Co., Ltd., TS: Nippon Beet Sugar Co., Ltd., CP: Commpro Co., Ltd., KD: Kurodaya Co., Ltd., KW: Kawadaen, HO: Honoka Ogre Farm, HE: HERBS & CROPS, SE: Eno Brostup S.A. , AL: Alisan Co., Ltd., VS: VSP Co., Ltd., AS: Amami Natural Science Research Institute Co., Ltd., EW: Everway Co., Ltd., CC: Chef's Choice Japan LLC, DH: Daihoku Co., Ltd., SN: Shimizu Nori Co., Ltd., HK: Saito Co., Ltd., Not specified: Home-grown The yield of A is indicated by the amount of defatted sample prepared from 50 g of plant body, and the yield of B is indicated by the weight of crude pectic polysaccharides prepared from 30 g of defatted sample (15 g of seaweed).

The catalytic activity and stereoselectivity of crude pectic polysaccharides prepared from aloe and almond by four different methods were measured using "Catalytic Activity Measurement Method I," and the results are shown together with the yields in Table 3. In the table, "A" indicates a defatted sample, while "B,""C,""D," and "E" indicate crude pectic polysaccharides prepared by the acetic acid method, hydrochloric acid method, EDTA method, and oxalic acid method, respectively.

By extracting crude pectic polysaccharides, it was found that plants from the following families, not described in Patent Documents 7, 8, and Non-Patent Document 11: Melastaceae, Malvaceae, Papayaceae, Moringaceae, Asteraceae, Lauraceae, Piperaceae, Houttuyniaceae, Lamiaceae, Oleaceae, Ebenaceae, Convolvulaceae, Amaranthaceae, Urticaceae, Moraceae, Myrtaceae, Fagaceae, Juglandaceae, Meliaceae, Aquifaceae, Rubiaceae, Xanthomonas, Palmaceae, Cupressaceae, and Equisetum, exhibited sufficient catalytic activity and stereoselectivity of 30% ee or greater. Furthermore, plants from the following families, Poaceae, Cucurbitaceae, Solanaceae, Rosaceae, Theaceae, and Amaryllidaceae, which exhibited low stereoselectivity, also exhibited stereoselectivity of 30% ee or greater. Furthermore, catalytic activity (specific activity per unit weight) was improved by up to 17-fold.

Example A2: Preparation of LP-LMW from Plant (Water-Soluble Soybean Polysaccharide) 7.5 g of water-soluble soybean polysaccharide (product name: SOYAFIBE S-DN, manufactured by Fuji Oil Co., Ltd.) was weighed out, 275 mL of purified water was added, and the mixture was sterilized in an autoclave (121°C, 15 minutes). A solution of 1.5 g of Lipase A "Amano" 6 (manufactured by Amano Enzyme Co., Ltd.), 15 mL of 0.5 M sodium phosphate buffer (pH 7), and 15 mL of purified water was added to the polysaccharide solution sterilized using a 0.45 μm filter and an autoclave, and the mixture was shaken at 37°C and 80 rpm for 20 hours. 40 mg of Proteinase K (manufactured by Takara Bio Inc.) was added to the shaken solution, and the mixture was further shaken for 2 hours. The pH of the reaction solution was then adjusted to 5 with acetic acid. A 0.5-fold volume of acetone was added to the pH-adjusted solution, and the mixture was stirred for 10 minutes using a stir bar. Insoluble matter was removed by centrifugation (10,000 × g, 10 minutes, 4 ° C). To the resulting supernatant, acetone (0.6 times the volume of the pH-adjusted solution) was added, and the mixture was stirred for 10 minutes using a stir bar. The resulting precipitate was then centrifuged (10,000 × g, 10 minutes, 4 ° C). The resulting precipitate was dissolved in purified water and sterilized in an autoclave (121 ° C, 15 minutes). This was centrifuged (15,000 × g, 10 minutes, 4 ° C) to remove insoluble matter. The resulting supernatant was placed in a dialysis tube (Visking tube, manufactured by Japan Medical Science Co., Ltd.) with a molecular weight cutoff (MWCO) of 12,000-14,000, and dialyzed using 10 mM acetic acid as the external solution. The liquid in the dialysis tube was freeze-dried, yielding 2.2 g of an LP-LMW fraction.

The catalytic activity, stereoselectivity, and average molecular weight of the main peak of water-soluble soybean polysaccharide (trade name: SOYAFIBE S-DN, manufactured by Fuji Oil Co., Ltd.) and the LP-LMW fraction were measured, and the sugar composition was analyzed. The results are shown in Tables 4 and 5. The catalytic activity and stereoselectivity were measured using "Catalytic Activity Measurement Method III," and the average molecular weight was measured using "Gel Filtration HPLC Analysis Method II."

Example A3: Degradation of Polysaccharides by Rhamnogalacturonate Lyase (RGL) (1) Purification of "Water-Soluble Soybean Polysaccharide Degrading Enzyme" (Lip-RGL) from a Commercially Available Enzyme 40 g of a commercially available enzyme (trade name: Lipase A "Amano" 6, manufactured by Amano Enzyme Co., Ltd.) was suspended in 10 mM potassium phosphate buffer (pH 7). Ammonium sulfate was added to the resulting suspension until a predetermined saturation percentage was reached. The precipitate formed from the ammonium sulfate fraction corresponding to 60% to 90% saturation was centrifuged and desalted by dialysis. The desalted liquid was applied to an anion exchange column (trade name: DEAE-Toyopearl, manufactured by Tosoh Corporation) and eluted with 0.2 M aqueous sodium chloride solution, and the resulting fraction was desalted.

The sample was applied to an FPLC system (AKTA explorer10S, GE Healthcare) equipped with a Mono Q 10-100 GL column (8.0 mL, GE Healthcare), and eluted using a gradient between buffer A (10 mM potassium phosphate buffer (pH 7)) and buffer B (10 mM potassium phosphate buffer (pH 7), 0.5 M sodium chloride). Fractions with enzyme activity were collected and desalted. The desalted solution was applied to an FPLC system (AKTA explorer 10S, GE Healthcare) equipped with a Mono Q 5-50 GL column (1.0 mL, GE Healthcare), and elution was performed by gradient elution between buffer A (10 mM potassium phosphate buffer (pH 7)) and buffer B (10 mM potassium phosphate buffer (pH 7), 0.5 M sodium chloride). Fractions that had enzyme activity and gave a single band on SDS-PAGE were collected, desalted, and used as purified water-soluble soybean polysaccharide-degrading enzyme (Lip-RGL).

Fractions from each purification step were subjected to SDS-PAGE to confirm the degree of purification. The results are shown in Figure 1. In Figure 1, lane 1 contains molecular weight markers, lane 2 contains a solution of a commercially available enzyme (Lipase A "Amano" 6, Amano Enzyme Co., Ltd.), lane 3 contains the ammonium sulfate fraction (60-90% saturated mixture), lane 4 contains the fraction eluted from a column (trade name: DEAE Toyopearl, Tosoh Corporation), lane 5 contains the fraction eluted from a column (trade name: Mono Q 10-100 GL, Sigma-Aldrich), and lane 6 contains the fraction eluted from a column (trade name: Mono Q 5-50, Sigma-Aldrich). The arrows in Figure 1 indicate the position of the band corresponding to purified Lip-RGL.

(2) Comparison of Enzyme Activity 25 mg of water-soluble soybean polysaccharide (trade name: SOYAFIBE S-DN, manufactured by Fuji Oil Co., Ltd.) was dissolved in 1 mL of 20 mM Bis-Tris HCl buffer (pH 6.5), and 50 μL of lipase (Lipase A "Amano" 6, manufactured by Amano Enzyme Co., Ltd.) solution or Lip-RGL solution was added. Each mixture was heated at 37°C for 2 hours and then filtered through a 0.45 μm membrane filter to obtain a filtrate. The molecular weight distribution of the resulting filtrate was evaluated using a TSKgel G5000PWXL column (manufactured by Tosoh Corporation) under the conditions of "Gel Filtration HPLC Analysis I." Comparison of chromatograms showed that degradation products with similar molecular weights were produced in both reactions, demonstrating that Lip-RGL can degrade water-soluble soybean polysaccharides.

Furthermore, when comparing the protein concentrations of the two enzyme solutions used, the protein concentration of the lipase (Lipase A "Amano" 6, manufactured by Amano Enzyme Co., Ltd.) solution was 2.6 mg/mL, while the protein concentration of the LipRGL solution was 0.026 mg/mL.

(3) Obtaining LipRGL gene and introducing it into Escherichia coli. Peptides prepared from LipRGL were subjected to nano LC-MS/MS analysis (device name: nanoACQUITY UPLC system and SYNAPT G2-Si High Definition Mass Spectrometer (both manufactured by Waters Corp.)) to deduce the amino acid sequences of the peptides.

According to Biotechnology and Bioprocess Engineering, 7, 57-66 (2002), the enzyme lipase A "Amano" 6 is derived from the black koji mold Aspergillus niger. The inventors then compared the obtained amino acid sequence information with entries from the genus Aspergillus (taxid: 5052) in the NCBI protein database, and extracted five enzymes (glucoamylase, cellobiohydrolase, rhamnogalacturonate lyase, endo-1,3-β-D-glucanase, and β-mannase) as candidates. Because this enzyme digests water-soluble soybean polysaccharides with a rhamnogalacturonan structure, they deduced that it was rhamnogalacturonate lyase.

(4) Peptide mapping of LipRGL
The amino acid sequence of rhamnogalacturonate lyase A (AkRGL) from Aspergillus luchuensis mut. kawachii NBRC4308 (GeneBank ID: GAA87164.1, SEQ ID NO: 1) has been published in the NCBI protein database. The amino acid sequence data of LipRGL obtained by nanoLC-MS/MS analysis closely matched the amino acid sequence of AkRGL. The results of peptide mapping of AkRGL and LipRGL are shown in Figure 2. Figure 2 shows the amino acid sequence of rhamnogalacturonate lyase A (AkRGL) from Aspergillus luchuensis mut. kawachii NBRC 4308. The underlined amino acids are the sequence derived from the LipRGL peptide.

The AkRGL gene information (GenBank ID: DF126458.1) was obtained from the NCBI gene database, and a gene for expression in E. coli (SEQ ID NO: 2) was artificially synthesized. Forward (SEQ ID NO: 3) and reverse (SEQ ID NO: 4) PCR primers were prepared based on the resulting gene. The gene was amplified by PCR using these primers and the synthetic AkRGL gene. The amplified gene was introduced into the pQE-80L vector (Qiagen) to obtain a plasmid (pQE80L-RGL) for expressing N-terminal His-tag-fused AkRGL. The resulting plasmid, pQE80L-RGL, was introduced into E. coli BL21(DE3), JM109, and Rosetta2(DE3), respectively, to prepare the transformants BL21(DE3)-AkRGL, JM109-AkRGL, and Rosetta2(DE3)-AkRGL. SDS-PAGE confirmed that each E. coli strain was able to produce AkRGL. GPC analysis also confirmed that the cell-free extract of each transformant could be used to prepare a catalyst (AkRGL-LMW) from SOYAFIBE S-DN, similar to lipase (Lipase A "Amano" 6, Amano Enzyme Co., Ltd.).

(5) Preparation of Recombinant RGL (Ak-RGL) Escherichia coli BL21(DE3)-AkRGL was cultured in 5 mL of LB medium (100 μg/mL ampicillin) at 37°C for 12 hours. The resulting culture was transferred to 500 mL of LB medium (100 μg/mL ampicillin) and cultured at 37°C for 9 hours with shaking at 150 rpm. After adding 1 mM IPTG, the culture was further cultured at 37°C for 12 hours. The E. coli cells were collected by centrifugation (8000 × g, 10 minutes, 4°C) and washed with 0.9% saline. The washed cells were suspended in 10 mM potassium phosphate buffer (pH 7) and disrupted with 0.1 mm diameter beads using a Multi-Beads Shocker (Yasui Kikai Co., Ltd.). Insoluble matter was removed by centrifugation (12000 × g, 15 minutes, 4°C) to obtain an extracellular extract.

The extracellular extract was applied to a Ni Sepharose 6 Fast Flow column (GE Healthcare Life Sciences, 3 x 8.5 cm), washed with 10 mM potassium phosphate buffer (pH 8.0) containing 0.1 M sodium chloride and 10 mM imidazole, and then eluted by successively increasing the imidazole concentration of the same buffer up to 300 mM. Enzyme-active fractions were pooled, desalted, and applied to an FPLC system (AKTA explorer 10S) equipped with a HisTrap HP column (GE Healthcare Life Sciences, 1.6 x 2.5 cm, 5 mL). After washing with 10 mM potassium phosphate buffer (pH 8.0) containing 0.1 M sodium chloride and 0.1 M imidazole, elution was performed by successively increasing the imidazole concentration of the same buffer up to 0.16 M. Only fractions with enzyme activity and showing a nearly single band on SDS-PAGE were pooled. The collected fractions were dialyzed against 1 mM potassium phosphate buffer (pH 7.0) to obtain purified AkRGL.

(6) Digestion of Soyafibe using recombinant RGL (AkRGL) Soyafibe S-DN (25 mg) was dissolved in 20 mM Bis-Tris-HCl buffer (pH 6.5, 1 mL), and 50 μL of either a lipase (Lipase A "Amano" 6, Amano Enzyme Co., Ltd.) solution, a purified LipRGL solution, or purified AkRGL was added. After heating at 37°C for 2 hours, the digestion solution was lyophilized to obtain a digest. The catalytic activity and stereoselectivity of the obtained digest were measured according to "Catalytic Activity Measurement Method II."

As shown in Table 6, both the digests produced by purified LipRGL and purified AkRGL had catalytic activity and stereoselectivity equivalent to those of SOYAFIBE S-DN. Furthermore, the catalytic activity of the digest produced by purified AkRGL (AkRGL-LMW) was 25.1 mU/mg.

Example A4: Decomposition with alkali (1) Preparation by sodium hydroxide and ethanol fractionation 100 g of water-soluble soybean polysaccharide (trade name: SOYAFIBE S-DN, manufactured by Fuji Oil Co., Ltd.) was added to an aqueous solution (2.5 L) of sodium hydroxide (200 g) and sodium borohydride (7.6 g), and the mixture was placed in a 3 L four-neck flask and heated in an oil bath with stirring to an internal temperature of 70°C for 120 hours. After neutralizing to pH 7 with acetic acid, insoluble matter was removed by centrifugation (12,000 × g, 20 minutes) to obtain a neutralized hydrolyzed solution, and its volume (X) was measured.

The neutralized hydrolysis solution was added dropwise to ethanol (0.5 volume of X) while stirring. The resulting precipitate was removed by centrifugation (12,000 x g, 20 minutes), and ethanol (0.3 volume of X) was added dropwise to the supernatant while stirring. The resulting precipitate was collected by centrifugation (12,000 x g, 20 minutes). The supernatant was used in the next step. The resulting precipitate was suspended in purified water and placed in a MWCO 3500 dialysis tube (BioDesign Dialysis Tubing, BioDesign Inc.) and dialyzed against 10 mM acetic acid solution as the external solution. The dialyzed solution was lyophilized to obtain 6.1 g of a polysaccharide fraction (AL-LMW EtOH 0.5/0.8).

While stirring the supernatant obtained in the previous step, ethanol (0.3 volume of X) was added dropwise. After the added solution was left to stand in a refrigerator for 3 days, the resulting precipitate was collected by centrifugation (12,000 x g, 20 minutes) (the supernatant was used in the next step). The resulting precipitate was suspended in purified water to a total volume of approximately 200 mL. The suspension was heated to approximately 40°C to dissolve the precipitate, and then stored in a refrigerator for 7 days. The precipitate that formed during refrigeration was collected by centrifugation (12,000 x g, 20 minutes). The resulting precipitate was suspended in 120 mL of purified water. The suspension was heated to approximately 40°C to dissolve the precipitate, then placed in a MWCO 3500 dialysis tube and dialyzed against 10 mM acetic acid solution as the external solution. The dialyzed solution was freeze-dried to obtain a 21.3 g fraction (AL-LMW EtOH 0.8/1.1 PPT). The remaining mother liquor, from which precipitates formed during refrigeration were collected, was placed in a dialysis tube with a MWCO of 3500 and dialyzed against 10 mM acetic acid solution as the external solution. The dialyzed solution was freeze-dried to obtain 1.9 g of a polysaccharide fraction (AL-LMW EtOH 0.8/1.1 SUP).

Ethanol (0.3 volume of X) was added dropwise to the supernatant obtained in the previous step while stirring. The added solution was left to stand in a refrigerator for one day, and the resulting precipitate was collected by centrifugation (12,000 × g, 20 minutes). The resulting precipitate was suspended in purified water, placed in a dialysis tube with a MWCO of 3500, and dialyzed against a 10 mM acetic acid solution as the external solution. The dialyzed solution was lyophilized to obtain a 1.4 g fraction (AL-LMW EtOH 1.1/1.4). The catalytic activity, stereoselectivity, and average molecular weight of each fraction were measured, and the results are shown in Table 7. Catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method III," and average molecular weight was measured according to "Gel Filtration HPLC Analysis Method II." The monosaccharide analysis results for each fraction are shown in Table 8.

(2) Preparation by Sodium Hydroxide and Acetone Fractionation 50 g of water-soluble soybean polysaccharide (trade name: SOYAFIBE S-DN) was added to 2.5 L of an aqueous solution containing 200 g of sodium hydroxide and 3.7 g of sodium borohydride, and the mixture was placed in a 3 L four-neck flask and heated in an oil bath with stirring to an internal temperature of 70°C for 102 hours. After neutralizing to pH 7 with acetic acid, insoluble matter was removed by centrifugation (12,000 × g, 20 minutes), and the volume (X) of the neutralized hydrolyzed solution was measured.

While stirring the neutralized hydrolysis solution, acetone (0.5 volume of X) was added dropwise. The resulting precipitate was collected by centrifugation (12,000 x g, 20 minutes) (the supernatant was used in the next step). The resulting precipitate was suspended in purified water, placed in a dialysis tube with a MWCO of 3500, and dialyzed against a 10 mM acetic acid solution. The dialyzed solution was freeze-dried to obtain 0.73 g of a polysaccharide fraction (AL-LMW Acetone 0/0.5).

While stirring the supernatant from the previous step, acetone (0.3 times the amount of acetone) was added dropwise. The resulting precipitate was collected by centrifugation (12,000 x g, 20 minutes) (the supernatant was used in the next step). The resulting precipitate was suspended in purified water, placed in a dialysis tube with a MWCO of 3500, and dialyzed against a 10 mM acetic acid solution as the external solution. The dialyzed solution was freeze-dried to obtain 10.7 g of a polysaccharide fraction (AL-LMW Acetone 0.5/0.8).

While stirring the supernatant obtained in the previous step, acetone (0.3 times the amount of X) was added dropwise. The resulting precipitate was collected by centrifugation (12,000 x g, 20 minutes). The resulting precipitate was suspended in purified water, placed in a dialysis tube with a MWCO of 3500, and dialyzed against a 10 mM acetic acid solution as the external solution. The dialyzed solution was freeze-dried to obtain 2.5 g of a polysaccharide fraction (AL-LMW Acetone 0.8/1.1).

The catalytic activity, stereoselectivity, and average molecular weight of each fraction were measured, and the results are shown in Table 9. Catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method III," and average molecular weight was measured according to "Gel Filtration HPLC Analysis Method II." The results of monosaccharide analysis of each fraction are shown in Table 10.

(3) Temperature Study: 4 g of water-soluble soybean polysaccharide (trade name: SOYAFIBE S-DN, manufactured by Fuji Oil Co., Ltd.) was added to 200 mL of an aqueous solution of sodium hydroxide (16 g) and sodium borohydride (0.30 g). 30 mL of the water-soluble soybean polysaccharide solution was placed in five 30 mm diameter test tubes and heated in a heat block to internal temperatures of 50, 60, 70, 80, and 90°C while stirring at 300 rpm. Eight mL of the same water-soluble soybean polysaccharide solution was placed in two 18 mm diameter test tubes, fitted with silicone stoppers, and heated at 121°C for 5 hours in an autoclave. Purified water (half the volume of the hydrolysis solution) was added to the hydrolysis solution, and the pH was adjusted to 6-7 with 50% acetic acid. After adjusting the pH, ethanol (three times the volume of the hydrolysis solution) was added dropwise, and the solution was allowed to stand in a refrigerator for 1 day. The resulting precipitate was collected by centrifugation (15,000 × g, 10 minutes). The resulting precipitate was suspended in purified water, placed in a dialysis tube with a MWCO of 3500, and dialyzed against a 10 mM acetic acid solution as the external solution. The dialyzed solution was centrifuged (15,000 × g, 10 minutes), and the supernatant was freeze-dried to obtain a polysaccharide fraction (AL-LMW).

The decomposition time, yield, catalytic activity, stereoselectivity, average molecular weight, and visual color of the hydrolyzed solution at the end of decomposition at each hydrolysis temperature are shown in Table 11. The catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method III," and the average molecular weight was measured according to "Gel Filtration HPLC Analysis Method II." As described above, hydrolyzates with catalytic activity were obtained by alkali treatment in the temperature range of 50 to 121°C.

(4) Study of Alkali Concentration: Water-soluble soybean polysaccharides (trade name: SOYAFIBE S-DN, manufactured by Fuji Oil Co., Ltd., 0.6 g) were added to an aqueous solution (30 mL) of sodium hydroxide (0.6 g) and sodium borohydride (45 mg) (final sodium hydroxide concentration: 0.5 M). Water-soluble soybean polysaccharide solutions were prepared in the same manner, except that the amount of sodium hydroxide was changed to 1.2 g, 1.8 g, 2.4 g, and 3.6 g, respectively (final sodium hydroxide concentrations: 1.0 M, 1.5 M, 2.0 M, and 3.0 M, respectively). Each solution was placed in a 30 mm diameter test tube and heated in a heat block to an internal temperature of 70°C while stirring at 300 rpm. The pH of the hydrolysis solution was adjusted to 6-7 with 50% acetic acid, and its volume (X) was measured. Ethanol (three times the amount of X) was added dropwise to the pH-adjusted solution, and the solution was allowed to stand in a refrigerator for 1 day. The resulting precipitate was collected by centrifugation (15,000 × g, 10 minutes). The resulting precipitate was suspended in purified water and placed in a dialysis tube with a MWCO of 3500. The suspension was dialyzed against a 10 mM acetic acid solution. The dialyzed solution was centrifuged (15,000 × g, 10 minutes), and the supernatant was freeze-dried to obtain a polysaccharide fraction (AL-LMW).

Table 12 shows the decomposition time, yield, catalytic activity, stereoselectivity, peak molecular weight, and color of the hydrolyzed solution at the end of decomposition for each alkali concentration. Catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method III," and average molecular weight was measured according to "Gel Filtration HPLC Analysis Method II." As described above, hydrolyzates with catalytic activity were obtained by alkali treatment in the alkali concentration range of 0.5 to 3.0 M.

(5) Preparation by potassium hydroxide and ethanol fractionation Four polysaccharide fractions (KOH-LMW-EtOH 0.5/0.8, KOH-LMW-EtOH 0.8/1.1-PPT, KOH-LMW-EtOH 0.8/1.1-SUP, and KOH-LMW-EtOH 1.1/1.4) were obtained in the same manner as in the above “(1) Preparation by sodium hydroxide and ethanol fractionation” except that 280.6 g of potassium hydroxide was used instead of sodium hydroxide.

The catalytic activity, stereoselectivity, and average molecular weight of each fraction are shown in Table 13. The catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method III," and the average molecular weight was measured according to "Gel Filtration HPLC Analysis Method II."

Example A5: Preparation of AL-LMW from "Okara" 5 g of okara powder (trade name: Okara Powder, manufactured by Acrosia Co., Ltd.) was suspended in 250 mL of an aqueous solution containing 20 g of sodium hydroxide and 0.38 g of sodium borohydride, placed in a 300 mL four-neck flask, and heated in an oil bath with stirring to an internal temperature of 70°C for 100 hours. The pH of the reaction solution was adjusted to 6 with acetic acid, and the hydrolyzed solution was centrifuged (12,000 x g, 15 minutes), and the volume (X) of the resulting supernatant was measured. Ethanol (0.5 times the amount of X) was added dropwise to the supernatant of the hydrolyzed solution while stirring, and the resulting precipitate was removed by centrifugation (12,000 x g, 15 minutes). Ethanol (0.3 times the amount of X) was added dropwise to the supernatant of this centrifuged solution, and the resulting precipitate was removed by centrifugation (12,000 x g, 15 minutes).

Ethanol (0.3 volume of X) was added dropwise to the supernatant of the centrifuged solution, and the resulting precipitate (EtOH 0.8/1.1 fraction) was obtained by centrifugation (12,000 x g, 15 minutes). Ethanol (0.3 volume of X) was also added dropwise to the supernatant of the centrifuged solution, and the resulting precipitate (EtOH 1.1/1.4 fraction) was obtained by centrifugation (12,000 x g, 15 minutes). Ethanol (0.6 volume of X) was added dropwise to the supernatant of the centrifuged solution, and the resulting precipitate (EtOH 1.4/2.0 fraction) was obtained by centrifugation (12,000 x g, 15 minutes). The resulting precipitates were each dissolved in purified water and stored in a refrigerator for 1-3 days. The precipitates that formed during refrigeration were centrifuged and separated into the precipitate (PPT) and the supernatant (SUP). The PPT portion was redissolved in purified water. The PPT solution and SUP were placed in a dialysis tube with a MWCO of 3500 and dialyzed against 10 mM acetic acid solution. The solution in the tube after dialysis was freeze-dried to obtain three fractions (EtOH 0.8/1.1, EtOH 1.1/1.4, EtOH 1.4/2.0) of PPT and SUP, respectively.

The catalytic activity, stereoselectivity, and average molecular weight of each fraction are shown in Table 14. Catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method III," and average molecular weight was measured according to "Gel Filtration HPLC Analysis Method II." Monosaccharide composition analysis was performed according to "Monosaccharide Composition Analysis Method," and the analysis results are shown in Table 15.

Example A6: Enzymatic Degradation of LP-LMW (1) Selection of Enzyme 50 mg/mL LP-LMW (100 μL), 100 mM potassium phosphate buffer (pH 7.0), and a commercially available enzyme solution (20 μL) listed in Table 16 were mixed in a 1.5 mL tube and reacted at 37°C and 1000 rpm for 22 hours. 20 μL of Proteinase K (Takara Bio Inc., Code No. 9034) was added to the reaction solution, and the mixture was further reacted at 37°C and 1000 rpm for 3 hours. After centrifugation, the supernatant was analyzed by "Gel Filtration HPLC Analysis Method I." When enzymes A, B, or E were used, degradation of LP-LMW with a molecular weight of approximately 40 kDa was confirmed, but when enzymes C or D were used, LP-LMW was not degraded. The reaction was performed in the same manner, varying the amount of enzyme A, B, or E added.

Figure 3 shows the results of time-dependent analysis under gel filtration HPLC analysis conditions I using 0.46 mU/mL of enzyme A. Figure 3 shows chromatograms of untreated (LP-LMW) and after 0.5, 1, 2, 4, 6, and 24 hours. Treatment with endo-β1,4-galactanases from different species resulted in degradation to a molecular weight of 3000 or less, suggesting that the main component of LP-LMW is β1,4-linked galactose.

(2) Fractionation of LP-LMW Using an Anion Exchange Resin Column LP-LMW (800 mg) was added to purified water (20 mL) and centrifuged to remove insoluble matter, including unwanted acidic sugars. The supernatant was loaded onto an OH-type anion exchange resin column (trade name: DEAE TOYOPEARL 650, manufactured by Tosoh Corporation, column volume (CV): 40 mL). After sample adsorption, 10 CV of purified water, 15 CV of 5 mM aqueous sodium bicarbonate, 5 CV of 10 mM aqueous sodium bicarbonate, and 5 CV of 100 mM aqueous sodium hydroxide were sequentially applied at a flow rate of approximately 3 mL/min, and fractionation was performed in 10 mL fraction volumes. The resulting fractions were subjected to measurement of total sugar content using the sulfuric acid-phenol method, and each eluted peak was fractionated and designated LP-LMW-D1 to D5 in order of elution. The resulting fractions were dialyzed and then lyophilized.

The yield, catalytic activity, stereoselectivity, and average molecular weight of each fraction are shown in Table 17. Catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method III," and average molecular weight was measured according to "Gel Filtration HPLC Analysis Method II." The results of the monosaccharide composition analysis are shown in Table 18.

(3) Partial Hydrolysis of LMW D1 Fraction (LP-LMW-D1) by Enzymatic Treatment: LP-LMW-D1 (343 mg) was weighed into a 200 mL Erlenmeyer flask and dissolved in purified water (32 mL) and 0.5 M MES buffer (pH 6.0, 8 mL) (final concentration: 10 mg/mL LMW, 100 mM MES buffer (pH 6.0)). Enzyme A (4 μL, final concentration: 1.8 mU/mL) solution was added to this substrate solution and shaken at 40°C and 140 rpm for 2 hours. Proteinase K (Takara Bio Inc., Code No. 9034, 20 μL) was added, and the mixture was incubated at 37°C and 1000 rpm for 30 minutes. The reaction was terminated by autoclaving. After cooling to room temperature, the solution was transferred to a 1000 MWCO dialysis tube and dialyzed against purified water. The dialyzed product was lyophilized to obtain the enzyme-treated product LP-LMW-D1deg (280 mg). The catalytic activity and stereoselectivity of the resulting fraction were measured according to "Catalytic Activity Measurement Method III," and the peak molecular weight was measured according to "Gel Filtration HPLC Analysis Method II." The results, along with the yield, are shown in Table 18.

(4) Fractionation of LP-LMW-D1deg using an anion exchange column
A sample prepared by dissolving LP-LMW-D1deg (280 mg) in purified water (2 mL) was applied to an OH-type anion exchange column (product name: DEAE TOYOPERAL 650, Tosoh Corporation, column volume (CV): 10 mL) equilibrated with purified water. After sample adsorption, 10 CV of purified water and 10 CV of 100 mM sodium bicarbonate solution were sequentially passed through the column using a peristaltic pump at a flow rate of approximately 3 mL/min, resulting in fractionation into 5 mL fractions. Of the sugar-containing fractions, the purified water-eluted fraction (LP-LMW-D1degD1) and the 100 mM sodium bicarbonate-eluted fraction (LP-LMW-D1degD2) were concentrated using an evaporator, transferred to a 1000 MWCO dialysis tube, and dialyzed against purified water. The dialyzed material was lyophilized, yielding 199 mg and 37.2 mg of each fraction, respectively.

The yield, catalytic activity, stereoselectivity, and average molecular weight of each fraction are shown in Table 19. Catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method III," and average molecular weight was measured according to "Gel Filtration HPLC Analysis Method II." The results of the monosaccharide composition analysis are shown in Table 20.

(5) Fractionation of LMW-D1degD1 by gel filtration column
A sample prepared by dissolving LMW-D1degD1 (150 mg) in purified water (0.5 mL) was applied to a gel filtration column (BioGel P10, Bio-Rad Laboratories, column volume (CV) 300 mL, internal diameter 3 cm × 54 cm). After the sample was adsorbed, 3 CV of 50 mM acetic acid solution was applied at a flow rate of approximately 1 mL/min, and fractionation was performed in 5 mL fractions.

The obtained fractions were analyzed according to "Gel filtration HPLC analysis method II" and were collected into five fractions based on molecular weight: more than 5 × 10 ³ , 4 to 5 × 10 ³ , 3 to 4 × 10 ³ , 2 to 3 × 10 ³ , and less than 2 × 10 ^3. Each fraction was lyophilized to obtain fractions BG1 (23.3 mg), BG2 (24.2 mg), BG3 (20.7 mg), BG4 (25.6 mg), and BG5 (33.2 mg), respectively.

The catalytic activity, stereoselectivity, average molecular weight, and monosaccharide analysis results of each fraction are shown in Table 21. Catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method III," average molecular weight according to "Gel Filtration HPLC Analysis Method II," and monosaccharide analysis according to "Monosaccharide Composition Analysis Method." Note that no sugars other than galactose and arabinose were detected in the monosaccharide analysis.

(6) Degradation of AL-LMW by Galactanase AL-LMW was purified using an anion exchange resin in the same manner as for LP-LMW to obtain AL-LMW-D1. The resulting AL-LMW-D1 was digested with Enzyme A (galactanase) and purified again using an anion exchange column to obtain DEAE-unadsorbed fractions (AL-LMW-D1degD1). The catalytic activity (measured by "Catalytic Activity Measurement Method III") and average molecular weight (measured by "Gel Filtration Analysis Method II") of SOYAFIBE S-DN, AL-LMW, AL-MLW-D1, and AL-LMW-D1degD1 are shown in Table 22. The monosaccharide composition of AL-LMW-D1degD1 was analyzed according to the "monosaccharide composition analysis method," and was found to be 0.2 mol% rhamnose, 0.3 mol% arabinose, 96.4% galactose, and 0.1 mol% galacturonic acid, with no other monosaccharides detected.

AL-LMW-D1degD1 was fractionated by gel filtration column chromatography to obtain fractions B1 to B4 in the order of first elution. The catalytic activity, stereoselectivity, and average molecular weight of each fraction are shown in Table 23. The catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method III," and the average molecular weight was measured according to "Gel Filtration HPLC Analysis II."

(7) Relationship between molecular weight and catalytic activity, and between molecular weight and stereoselectivity
The relationships between molecular weight, catalytic activity, and stereoselectivity for fractions obtained by digesting or purifying LP-LMW and AL-LMW are summarized in Figure 4. A tendency for catalytic stereoselectivity to drop sharply was observed when the molecular weight was between 2.5 x ¹⁰ and 3 x ^10. A tendency for it to improve with molecular weight was observed when the molecular weight was between ^2.5 x 10 and 6 x ¹⁰ .

Example A7: Hydrolysis of polysaccharides with acid (1) Investigation of acidic conditions 5 g of the polysaccharide fraction (AL-LMW EtOH 0.8/1.1-PPT) was dissolved in purified water to obtain 500 mL of an AL-LMW EtOH 0.8/1.1-PPT solution.

30 mL of AL-LMW EtOH 0.8/1.1-PPT solution was dispensed into beakers and adjusted to pH 2, 2.5, 3.5, and 4 with dilute sulfuric acid. The solution was then transferred to test tubes and heated in a heat block to an internal temperature of 90°C while stirring. After hydrolysis had progressed, 3.8 mL of sample was taken from each test tube, and 0.2 mL of 1 M sodium acetate solution (pH 5) and 12 mL of ethanol were added sequentially. The mixture was then stored in a refrigerator for one day. The resulting precipitate was collected by centrifugation (15,000 x g, 10 minutes) and air-dried. The dried precipitate was suspended in a small amount of purified water and lyophilized to obtain the low molecular weight polysaccharide fraction.

The AL-LMW EtOH 0.8/1.1-PPT solution was adjusted to pH 3 with dilute sulfuric acid, and 30 mL of each was placed in test tubes and heated at an internal temperature of 70°C, 80°C, or 90°C while stirring. After hydrolysis had progressed, 3.8 mL of a sample was taken from each test tube, and 0.2 mL of 1 M sodium acetate solution (pH 5) and 12 mL of ethanol were added sequentially, followed by storage in a refrigerator for one day. The resulting precipitate was collected by centrifugation (15,000 x g, 10 minutes) and air-dried. The dried precipitate was suspended in a small amount of purified water and lyophilized to obtain the low molecular weight polysaccharide fraction.

Dilute sulfuric acid was added to the AL-LMW EtOH 0.8/1.1-PPT solution to adjust the pH to 3, and 7 mL of this solution was transferred to a test tube, fitted with a silicone stopper, and heated to 121°C in an autoclave. After hydrolysis had progressed, 0.25 mL of 1M sodium acetate solution (pH 5) and 15 mL of ethanol were added sequentially to 4.75 mL of a sample collected from the test site, and the mixture was stored in a refrigerator for one day. The resulting precipitate was collected by centrifugation (15,000 x g, 10 minutes) and air-dried. The dried precipitate was suspended in a small amount of purified water and freeze-dried to obtain a low molecular weight polysaccharide fraction.

Table 24 shows the hydrolysis time under each hydrolysis condition, the yield of the obtained low-molecular-weight polysaccharide fraction, and the catalytic activity, stereoselectivity, and average molecular weight of the obtained hydrolysis product. Catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method III," and average molecular weight was measured according to "Gel Filtration HPLC Analysis II." The yields in Table 24 are calculated based on the amount of crude product obtained by ethanol precipitation, and therefore contain inorganic salts, and may exceed 100%. It was confirmed that hydrolysis with acid proceeded at temperatures ranging from 70 to 121°C and pH values ranging from 2 to 4.

(2) Hydrolysis with Sulfuric Acid: The fraction AL-LMWEtOH0.8/1.1 PPT (17 g) obtained above was placed in a beaker, and purified water (800 mL) was added and stirred to dissolve. 1 M sulfuric acid was added to adjust the pH of the solution to 3.2, and purified water was added to bring the total volume to 850 mL. The resulting solution was transferred to a 1 L four-neck flask, fitted with a reflux condenser, and stirred in an oil bath at an internal temperature of 90 °C for 88 hours.

Then, the pH was adjusted to 6-7 with 1 M sodium hydroxide, and the solution was centrifuged (12,000 x g, 20 minutes) to remove insoluble matter, obtaining a neutralized hydrolyzed solution, and its volume (X) was measured.

While stirring, ethanol (same amount as X) was added dropwise to the neutralized hydrolysis solution. The resulting precipitate was collected by centrifugation (12,000 x g, 20 minutes) (the separated supernatant 1 was used in the next step). The resulting precipitate was suspended in purified water, placed in a dialysis tube with a MWCO of 1000, and dialyzed against a 10 mM acetic acid solution as the external solution. The dialyzed solution was freeze-dried to obtain 1.2 g of a low molecular weight polysaccharide fraction (AS-LLMW EtOH 0/1.0).

Supernatant 1 was cooled in an ice-water bath, and ethanol (same amount as X) was added dropwise and stirred. The resulting precipitate was collected by centrifugation (12,000 x g, 20 minutes). (The separated Supernatant 2 was used in the next step.) The resulting precipitate was suspended in purified water, placed in a 1000 MWCO dialysis tube, and dialyzed against 10 mM acetic acid solution as the external solution. The dialyzed solution was lyophilized to yield 6.2 g of acid-treated low molecular weight polysaccharide fraction (AS-LLMW EtOH 1.0/2.0).

Ethanol (same amount as X) was added dropwise to Supernatant 2 and stirred. The resulting precipitate was collected by centrifugation (12,000 x g, 20 minutes) (Separated Supernatant 3 was used in the next step). The resulting precipitate was suspended in purified water, placed in a 1000 MWCO dialysis tube, and dialyzed against 10 mM acetic acid solution as the external solution. The dialyzed solution was lyophilized to yield 0.96 g of low molecular weight polysaccharide fraction (AS-LLMW EtOH 2.0/3.0).

The catalytic activity, stereoselectivity, and peak molecular weight of each fraction are shown in Table 25. Catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method III," and average molecular weight was measured according to "Gel Filtration HPLC Analysis Method II." Monosaccharide composition analysis was performed according to "Monosaccharide Composition Analysis Method," and the results are shown in Table 26.

(3) Hydrolysis using a cation exchange resin. The fraction (AL-LMW EtOH 0.8/1.1 PPT) (9.0 g) was placed in a beaker, purified water (900 mL) was added, and the mixture was stirred to dissolve. The mixture was then poured into a 500 mL four-neck flask. Regenerated Amberlyst 15R (equivalent to 10.8 g of dry Amberlyst) was added to the flask, a reflux condenser was attached, and the mixture was heated in an oil bath and stirred at an internal temperature of 90°C for 103 hours. The pH of the solution at this point was 3.4.

The pH was then adjusted to 6-7 with 1M aqueous sodium hydroxide, and the solution was centrifuged (12,000 x g, 20 minutes) to remove insoluble matter, yielding a neutralized hydrolysis solution whose volume (X) was measured. Ethanol (same volume as X) was added dropwise to the neutralized hydrolysis solution while stirring. The resulting precipitate was removed by centrifugation (12,000 x g, 20 minutes). Ethanol (0.4 volume of X) was added dropwise to Supernatant 1 while stirring. The resulting precipitate was collected by centrifugation (12,000 x g, 20 minutes). (The separated Supernatant 2 was used in the next step.) The resulting precipitate was suspended in purified water, placed in a 1000 MWCO dialysis tube, and dialyzed against a 10 mM acetic acid solution as the external solution. The dialyzed solution was freeze-dried to yield 2.6 g of a low molecular weight polysaccharide fraction (AM-LLMW EtOH 1.0/1.4).

Supernatant 2 was cooled in an ice-water bath and stirred while ethanol (0.6 volume of X) was added dropwise. The resulting precipitate was collected by centrifugation (12,000 x g, 20 minutes) (the separated supernatant 3 was used in the next step). The resulting precipitate was suspended in purified water, placed in a 1000 MWCO dialysis tube, and dialyzed against 10 mM acetic acid solution as the external solution. The dialyzed solution was lyophilized to yield 2.0 g of low molecular weight polysaccharide fraction (AM-LLMW EtOH 1.4/2.0).

While stirring the supernatant 3, ethanol (same amount as X) was added dropwise. The resulting precipitate was collected by centrifugation (12,000 x g, 20 minutes). The resulting precipitate was suspended in purified water, placed in a dialysis tube with a MWCO of 1000, and dialyzed against a 10 mM acetic acid solution as the external solution. The dialyzed solution was lyophilized to obtain 0.52 g of a low molecular weight polysaccharide fraction (AM-LLMW EtOH 2.0/3.0).

The catalytic activity, stereoselectivity, and average molecular weight of each fraction are shown in Table 27. The catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method III," and the average molecular weight was measured according to "Gel Filtration HPLC Analysis Method II."

Table 28 shows the results of "monosaccharide composition analysis" of fractions prepared in the same manner but with different ratios of ethanol added after hydrolysis.

(4) Hydrolysis with Acetic Acid: The fraction (AL-LMW EtOH 0.8/1.1 PPT) (0.9 g) was placed in a beaker, and purified water (90 mL) was added. The mixture was stirred and dissolved. The pH of the solution was adjusted to 3.1 with 1 M acetic acid. The solution was transferred to a 100 mL four-neck flask, fitted with a reflux condenser, heated in an oil bath, and stirred at an internal temperature of 90°C for 88 hours. The pH was then adjusted to 5 with 1 M sodium hydroxide to obtain a neutralized hydrolysis solution, and its volume (X) was measured. Ethanol (the same amount as X) was added dropwise while stirring the neutralized hydrolysis solution. The resulting precipitate was removed by centrifugation (15,000 × g, 10 minutes), and ethanol (0.4 volume of X) was added dropwise to the supernatant while stirring. The resulting precipitate was collected by centrifugation (12,000 × g, 20 minutes) (the separated supernatant 1 was used in the next step). The resulting precipitate was suspended in purified water, placed in a dialysis tube with a MWCO of 1000, and dialyzed against a 10 mM acetic acid solution as the external solution. The dialyzed solution was freeze-dried to obtain 0.17 g of an acid low molecular weight fraction (AA-LLMW EtOH 1.0/1.4).

While stirring Supernatant 1, ethanol (0.6 volume of X) was added dropwise. The resulting precipitate was collected by centrifugation (15,000 x g, 10 minutes) (the separated Supernatant 2 was used in the next step). The resulting precipitate was suspended in purified water, placed in a 1000 MWCO dialysis tube, and dialyzed against 10 mM acetic acid solution as the external solution. The dialyzed solution was lyophilized to obtain 0.15 g of acid-treated low molecular weight fraction (AA-LLMW EtOH 1.4/2.0).

While stirring the supernatant 2, ethanol (same amount as X) was added dropwise. The resulting precipitate was collected by centrifugation (15,000 x g, 10 minutes). The resulting precipitate was suspended in purified water, placed in a dialysis tube with a MWCO of 1000, and dialyzed against a 10 mM acetic acid solution. The dialyzed solution was freeze-dried to obtain 0.032 g of the acid-treated low molecular weight fraction (AA-LLMW EtOH 2.0/3.0).

The catalytic activity, stereoselectivity, and average molecular weight of each fraction are shown in Table 29. The catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method III," and the average molecular weight was measured according to "Gel Filtration HPLC Analysis Method II."

(5) Purification by anion exchange resin. The fraction (AM-LLMW EtOH 1.4/2.0) (3 g) was suspended in purified water (100 mL) and placed in a 1000 MWCO dialysis tube (Spectra/Por 6 Dialysis Membrane, manufactured by REPLIGEN). Dialysis was performed using purified water as the external solution. The dialyzed solution (2.7 g of catalyst) was applied to an anion exchange column (trade name: DEAE-Toyopearl (OH type), manufactured by Tosoh Corporation, column volume: 280 mL), and purified water was passed through at a flow rate of approximately 8 mL/min. Fractionation was performed at a fraction volume of 16 mL. Fractions containing a large amount of sugar were collected, placed in a 1000 MWCO dialysis tube, and dialyzed against 10 mM acetic acid as the external solution. The dialyzed solution was lyophilized to obtain a DEAE-unadsorbed fraction (AM-LLMW EtOH 1.4/2.0 D1(FT)) (1.87 g, yield: 71%).

The catalytic activity and stereoselectivity of the DEAE-unadsorbed fraction were measured according to "Catalytic Activity Measurement Method III," and the peak molecular weight was measured according to "Gel Filtration HPLC Analysis Method II." The catalytic activity was 17.2 mU/mg, the stereoselectivity was 66.8% ee, and the peak molecular weight was 3.9 × 10 ^3. Furthermore, when monosaccharide composition analysis was performed according to "Monosaccharide Composition Analysis Method," this fraction was composed of 0.1% fructose, 3.1% arabinose, and 96.8% galactose, with no other sugars detected.

(6) Purification by Gel Filtration Column The DEAE-unadsorbed fraction (300 mg) obtained above was dissolved in purified water to obtain 1 mL of aqueous solution. This solution was loaded onto a BioGel P10 column (Bio-Rad Laboratories, 300 mL column capacity, 3 cm inner diameter x 54 cm). After sample adsorption, a 50 mM acetic acid solution was applied at a flow rate of approximately 1 mL/min, and 3 CV fractions were obtained with a fraction volume of 5 mL.

The obtained fractions were analyzed by "Gel filtration HPLC analysis II" to measure the molecular weight and relative sugar content. Fractions with molecular weights of more than 4.5 × 10 ³ , 3.5 to 4.5 × 10 ³ , and less than 3.5 × 10 ³ were collected, and then fractions equivalent to three column volumes were combined by gel filtration chromatography and lyophilized to obtain fractions BG1 to BG3.

The catalytic activity, stereoselectivity, and average molecular weight of each fraction are shown in Table 30. Catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method III," and average molecular weight was measured according to "Gel Filtration HPLC Analysis Method II."

Example A8: Preparation from Carrots (1) Preparation of Pectic Polysaccharides from Carrots Approximately 50 g of powdered carrot (product name: Carrot Fine Powder, manufactured by Mikasa Sangyo Co., Ltd.) was weighed into a 500 mL Erlenmeyer flask, and 300 mL of a mixture of chloroform and methanol (volume ratio 9:1) was added. The flask was covered with aluminum foil and vigorously stirred for 1 hour using a stirrer. After suction filtration using a suction funnel and suction bottle, the flask and filtered plant matter were washed with the same mixture and recovered. The recovered plant matter and 300 mL of the same mixture were placed in a flask, stirred again for 1 hour using a stirrer, and then recovered by filtration. This process was repeated twice (total of 3 times) to obtain 45 g of a powdered defatted sample.

4 g of the defatted sample was suspended in 30 mL of purified water, stirred in a refrigerator for one day, heated at 50°C for one hour, and then centrifuged (15,000 x g, 15 minutes, 4°C) to obtain a precipitate. The resulting precipitate was suspended in 40 mL of 10 mM acetic acid, heated at 121°C for one hour using an autoclave, and then centrifuged (15,000 x g, 15 minutes, 4°C) to obtain the supernatant, and its volume (X) was measured. Ethanol (twice the volume of X) was added dropwise to the resulting supernatant while stirring, and the resulting precipitate was collected by centrifugation. This precipitate was placed in a desiccator and dried under reduced pressure to obtain 0.303 g of powder (crude pectic polysaccharides).

(2) Purification of Carrot-Derived Pectic Polysaccharides Pectic polysaccharides (238 mg) were suspended in an aqueous solution (27 mL) of sodium hydroxide (1.08 g) and sodium borohydride (41 mg) and placed in a test tube. The suspension was heated using an aluminum block, stirred at an internal temperature of 70°C for 40 hours, and neutralized to pH 6 with acetic acid to obtain a neutralized hydrolyzed solution, the volume of which (X) was measured. Ethanol (0.5 volume of X) was added dropwise to the neutralized hydrolyzed solution while stirring. The resulting precipitate was removed by centrifugation (15,000 × g, 10 minutes), and ethanol (0.6 volume of X) was added dropwise to the supernatant while stirring, followed by standing in a refrigerator for one day. The resulting precipitate was collected by centrifugation (15,000 × g, 10 minutes), dissolved in purified water (1.3 mL), and subjected to a desalting column (product name: PD-10). The sugar-containing eluate was collected and freeze-dried to obtain 87 mg of a fraction (AL-LMW EtOH 0.5/1.1).

The catalytic activity, stereoselectivity, and average molecular weight of each fraction are shown in Table 31. The catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method III," and the average molecular weight was measured according to "Gel Filtration HPLC Analysis Method II."

Compared to the raw material powdered carrot, the catalytic activity of crude pectic polysaccharide was 3.9 times higher, and that of the fraction (AL-LMW EtOH 0.5/1.1) was 6.1 times higher. The optical purity of the amino alcohol produced by the epoxy ring-opening reaction was 53% ee for powdered carrot, 64% ee for crude pectic polysaccharide, and 66% ee for the fraction (AL-LMW EtOH 0.5/1.1), demonstrating improved stereoselectivity.

(3) Preparation of Polysaccharide Fraction and Low Molecular Weight Polysaccharide Fraction from Plant Processed Product (Powdered Carrot) Powdered carrot (product name: Ninjin Powder, manufactured by Kodama Foods Co., Ltd.) (18 g) was suspended in an aqueous solution (925 mL) of sodium hydroxide (72 g) and sodium borohydride (1.36 g), placed in a 1 L four-neck flask, heated in an oil bath, and stirred at an internal temperature of 70°C for 122 hours. The reaction solution was neutralized to pH 7 with acetic acid to obtain a neutralized hydrolyzed solution, and its volume (X) was measured. Ethanol (0.5 volume of X) was added dropwise to the neutralized hydrolyzed solution while stirring. The resulting precipitate was removed by centrifugation (12,000 × g, 20 minutes), and ethanol (0.6 volume of X) was added dropwise to the resulting supernatant while stirring, followed by standing in a refrigerator for one day. The resulting precipitate was collected by centrifugation (12,000 × g, 20 minutes), suspended in purified water, and placed in a dialysis tube with a MWCO of 3500. The external solution was dialyzed against a 10 mM acetic acid solution. The dialyzed solution was lyophilized to obtain 0.28 g of a polysaccharide fraction (AL-LMW EtOH 0.5/1.1).

AL-LMW (240 mg) was suspended in purified water (24 mL), and the pH of the solution was adjusted to 3 with 1 M sulfuric acid. The solution was placed in a test tube and stirred for 96 hours while being heated with an aluminum block to maintain an internal temperature of 90°C. The volume of the hydrolyzed solution was measured, and a 0.053-fold equivalent amount of 1 M sodium acetate (pH 5) was added and stirred (final concentration 50 mM). The solution was then centrifuged (15,000 x g, 15 minutes) to obtain a neutralized hydrolyzed solution (supernatant), and its volume (X) was measured.

Ethanol (0.5 volume of X) was added dropwise to the neutralized hydrolysis solution while stirring. The resulting precipitate was removed by centrifugation (15,000 x g, 20 minutes). Ethanol (0.5 volume of X) was added dropwise to the resulting supernatant while stirring. This procedure was repeated twice (3 times in total). The resulting precipitate was removed by centrifugation (15,000 x g, 20 minutes). Ethanol (the same volume as X) was added dropwise to the resulting supernatant while stirring. After leaving the solution in the refrigerator for one day, the resulting precipitate was collected by centrifugation (15,000 x g, 15 minutes). The resulting precipitate was dissolved in 1 mL of purified water and centrifuged (15,000 x g, 10 minutes). The supernatant was applied to a desalting column (product name: PD-10) to obtain an eluate containing sugars. This eluate was applied to a column (product name: DEAE-Toyopearl (OH type), Tosoh Corporation, column volume: 4 mL) and purified water was used. The unadsorbed fractions containing sugars were collected and freeze-dried to obtain 10.7 mg of an acid-decomposed low molecular weight polysaccharide fraction (AS-LLMW EtOH 1.0/2.0 DEAE-FT).

The catalytic activity, stereoselectivity, and average molecular weight of each fraction are shown in Table 32. The catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method III," and the average molecular weight was measured according to "Gel Filtration HPLC Analysis Method II."

Compared to the raw material carrot powder, the catalytic activity of the polysaccharide fraction (AL-LMW EtOH 0.5/1.1) was 2.3 times higher, and that of the acid-degraded low molecular weight polysaccharide fraction (AS-LLMW EtOH 1.0/2.0 DEAE-FT) was 9.4 times higher. The optical purity of the amino alcohols produced by the epoxy ring-opening reaction was 41% ee for carrot powder, 56% ee for the polysaccharide fraction (AL-LMW EtOH 0.5/1.1), and 64% ee for the acid-degraded low molecular weight polysaccharide fraction (AS-LLMW EtOH 1.0/2.0 DEAE-FT), demonstrating improved stereoselectivity.

Example A9: Preparation of a Low-Molecular-Weight Polysaccharide Fraction Directly from Water-Soluble Soybean Polysaccharides (1) Fractionation of Water-Soluble Soybean Polysaccharides Using an Anion Exchange Column SOYAFIVE S-DN (Fuji Oil Co., Ltd., 4 g) was dissolved in purified water (40 mL), autoclaved, and then centrifuged to remove insoluble matter. The supernatant was applied to a column (trade name: DEAE TOYOPERAL 650 (OH type), Tosoh Corporation, column volume (CV) 160 mL) equilibrated with 10 mM aqueous sodium bicarbonate. After sample adsorption, a peristaltic pump was used to sequentially pass 5 CV of 10 mM aqueous sodium bicarbonate, 5 CV of 100 mM aqueous sodium bicarbonate, 5 CV of 100 mM aqueous sodium carbonate, and 5 CV of 300 mM aqueous sodium carbonate at a flow rate of approximately 5 mL/min, resulting in fractionation at a volume of 16 mL. The resulting fractions were each loaded onto a TLC column (1 μL), which was then immersed in 10% sulfuric acid and heated to identify the sugar-containing fractions. The fractions eluted with 10 mM and 100 mM aqueous sodium bicarbonate were designated SFD1 and SFD2, respectively, while the fractions eluted with 100 mM and 300 mM aqueous sodium carbonate were designated SFD3 and SFD4, respectively. Each fraction was concentrated using an evaporator, then transferred to a dialysis tube with a MWCO of 12,000-14,000 and dialyzed in purified water. The dialyzed solution was lyophilized, and the catalytic activity of SFD1-4 was measured according to "Catalytic Activity Measurement Method IV." The results are shown in Table 33.

(2) Partial Degradation of Fraction SFD2/3 by Enzyme Treatment The mixture of SFD2 and SFD3 obtained above is referred to as "SFD2/3." 100 mL of 50 mM sodium acetate buffer (pH 5.0) containing SFD2/3 (20 mg) was prepared. This solution was placed in a 500 mL Erlenmeyer flask, and enzyme A solution (8 μL, final concentration: 14.8 mU/mL) was added. The mixture was then shaken at 37°C and 140 rpm for 2 hours. The reaction was then stopped by heating in a 142°C oil bath and stirring for 10 minutes. After cooling to room temperature, purified water was added to the mixture and the volume was adjusted to 100 mL. Ethanol (150 mL) was added dropwise while stirring the solution. After stirring for an additional 10 minutes, the mixture was centrifuged (12,000 × g, 10 minutes, 4°C) to obtain a precipitate (SFD2/3degE1) and a supernatant (SFD2/3degE2). SFD2/3degE1 was dissolved in purified water. The ethanol in SFD2/3degE2 was removed using an evaporator. Each solution was transferred to a dialysis tube with a MWCO of 1000 and dialyzed against purified water. After lyophilization, SFD2/3degE1 and SFD2/3degE2 were obtained as powders. The yields were 79% and 12%, respectively.

(3) Fractionation of SFD2/3degE2 by an anion exchange column. Ten mL of a 100 mg/mL SFD2/3degE2 solution was applied to a column (DEAE TOYOPERAL 650 (OH type), Tosoh Corporation, column volume (CV): 40 mL) equilibrated with purified water. After sample adsorption, 10 CV of purified water and 10 CV of 100 mM sodium carbonate aqueous solution were sequentially passed through the column at a flow rate of approximately 3 mL/min using a peristaltic pump, and fractionation was performed in 10 mL fraction volumes. Of the sugar-containing fractions, the fraction eluted with purified water was designated SFD2/3degE2D1 (the first half of the fraction was designated SFD2/3degE2D1-1, and the second half was designated SFD2/3degE2D1-2), and the fraction eluted with 100 mM sodium carbonate aqueous solution was designated SFD2/3degE2D2. Each fraction was concentrated using an evaporator, transferred to a dialysis tube with a MWCO of 1000, dialyzed against purified water, and freeze-dried.

The catalytic activity, stereoselectivity, peak molecular weight, and monosaccharide analysis results for each fraction are shown in Table 34. Catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method III," average molecular weight according to "Gel Filtration HPLC Analysis Method II," and monosaccharide analysis according to "Monosaccharide Composition Analysis Method." The catalytic activity of SFD2/3degE2D1 was 15 mU/mg.

(4) Fractionation of SFD2/3degE2D1 by Gel Filtration Column. A 300 mg/mL aqueous solution of SFD2/3degE2D1 (1 mL) was applied to an open column (BioGel P10, Bio-Rad Laboratories, column volume (CV) 300 mL) equilibrated with 50 mM acetic acid. After sample adsorption, 50 mM acetic acid solution was applied at a flow rate of approximately 1 mL/min, and 3 CV fractions were obtained with a fraction volume of 5 mL. Each fraction was analyzed under gel filtration HPLC analysis II conditions, and the estimated molecular weight and peak size were measured. Fractions with a molecular weight range of approximately 1 x 10 ³ were collected and lyophilized to obtain multiple neutral galactan fractions (fraction NG).

The catalytic activity, stereoselectivity, average molecular weight, and arabinose content of each fraction NG are shown in Figures 5 and 6. Catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method III," average molecular weight according to "Gel Filtration HPLC Analysis Method II," and arabinose content according to "Monosaccharide Composition Analysis Method." Figures 5(a) and 5(b) are graphs plotting the estimated molecular weight and catalytic activity of the neutral galactan fractions, respectively. When the estimated molecular weight was 3 x 10 ³ or higher, the catalytic activity tended to increase with increasing estimated molecular weight. The catalytic activity plateaued around 6 x 10 ^3. When the estimated molecular weight was 3,000 or less, the stereoselectivity of the catalyst was found to decrease significantly. The results of the monosaccharide composition analysis are shown in Figure 6. As shown in Figure 6, the arabinose content tended to increase with increasing estimated molecular weight. The arabinose content was approximately 5% for fractions with estimated molecular weights of approximately 2.5 x 10 ³ to 7 x 10 ^3.

Example A10: Purification of low molecular weight polysaccharide fraction (NG) using HILIC-HPLC (1) Degree of polymerization estimation by HILIC-HPLC analysis An oligosaccharide (synthetic Gal9) consisting of β1,4-linked galactose with a degree of polymerization (DP) of 9 was synthesized according to the literature (Chemistry A European Journal, 22, 11543-11548 (2016), European Journal of Organic Chemistry, 3849-3869 (2001)). The catalytic activity of synthetic Gal9 was approximately 0.4 mU/mg, which was comparable to that of commercially available D-galactose, showing almost no catalytic activity, and no stereoselectivity was confirmed.

The fraction prepared in Example A9 (estimated molecular weight: 1 to 2×10 ³ ) was analyzed under the following HILIC analysis conditions.

<HILIC-HPLC analysis conditions>
Column: SugarD (5 μm, 4.6 mm I.D. × 250 mm, manufactured by Nacalai Tesque)
Eluent: acetonitrile/purified water = 65/35
Flow rate: 1.0 ml/min Column temperature: 30°C
Sample concentration: 1 mg/mL
Injection volume: 10μL
Detector: RID

The chromatogram exhibited a sawtooth pattern, with multiple alternating peaks detected at high sensitivity (major peaks) and peaks detected at low sensitivity (minor peaks). Because the synthetic Gal9 peak overlapped with one of the major peaks, this peak was designated the "9mer." Using this peak as the base, the major peaks were numbered with integers in the order in which they eluted. Minor peaks sandwiched between major peaks were numbered by adding ".5" to the number of the major peak that eluted immediately before them. For example, the minor peak eluting immediately before the "9mer" (synthetic Gal9) was the "8.5mer," and the major peak eluting immediately before the "8.5mer" was the "8mer."

(2) HILIC-HPLC Separation of "Fraction NG3-4 kDa" The fraction with a molecular weight of 3-4 x ¹⁰³ (fraction NG3-4 kDa) obtained in Example A9 was separated under the following HILIC separation conditions. The sample was dissolved in purified water to a concentration of 300 mg/mL, and the supernatant was obtained by centrifugation and filtered through a 0.2 μm filter. The corresponding fractions were mixed, the acetonitrile was removed using an evaporator, and the mixture was lyophilized. The obtained fractions were analyzed under the HILIC-HPLC analysis conditions described in (1) above, except that the eluent was changed to acetonitrile/purified water = 62/38, and each peak was numbered in the same manner.

<HILIC-HPLC preparative conditions>
Column: SugarD (5 μm, 20 mm I.D. × 250 mm, manufactured by Nacalai Tesque) and guard column eluent: acetonitrile/purified water = 60/40
Flow rate: 10 ml/min Column temperature: 30°C
Sample concentration: 1 mg/mL
Injection volume: 100μL (=30mg)
Fraction volume: 10 mL
Detector: RID

The catalytic activity, stereoselectivity, and monosaccharide composition analysis results for each fraction are shown in Table 35. Catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method IV (0.1 mg catalyst amount)," and monosaccharide composition analysis was measured according to "Monosaccharide Composition Analysis Method." Figure 7(a) is a chromatogram of the HILIC-HPLC preparative fraction of "Fraction NG 3-4 kDa," and Figure 7(b) is a graph showing an overlay of chromatograms obtained by HILIC-HPLC analysis of each fraction after preparative separation. Each fraction showed a single peak in HPLC analysis.

(3) HILIC-HPLC Separation of "Fraction NG 2-3 kDa" The fraction with a molecular weight of 2-3 x ¹⁰³ (fraction NG 2-3 kDa) obtained in Example A9 was subjected to HILIC-HPLC separation under the HILIC analysis conditions described in (2) above, except that the eluent was changed to acetonitrile/purified water (63/37).

The major peak fraction was designated "pool a" and the minor peak fraction was designated "pool b," and the two were combined. The acetonitrile was removed using an evaporator and the mixture was freeze-dried. The resulting pool a and b fractions were dissolved in purified water to a concentration of 150 mg/mL, centrifuged to obtain the supernatant, filtered through a 0.2 μm filter, and then separated again under the above conditions. The corresponding fractions were combined, the acetonitrile was removed using an evaporator, and the mixture was freeze-dried. The resulting fractions were analyzed under the HILIC-HPLC analysis conditions described above (1), except that the eluent was changed to acetonitrile/purified water (62/38), and each peak was numbered.

The catalytic activity, stereoselectivity, and monosaccharide composition analysis results for each fraction are shown in Table 36. Catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method IV (0.1 mg catalyst amount)," and monosaccharide composition analysis was measured according to "Monosaccharide Composition Analysis." Figure 8(a) shows a chromatogram of "Fraction NG 2-3 kDa" purified once by HILIC-HPLC preparative separation. Figure 8(b) shows chromatograms of "Pool a" and "Pool b," respectively, purified by HILIC-HPLC preparative separation. Figure 8(c) is a graph showing an overlay of chromatograms of the HILIC-HPLC analysis of each fraction after separation. Each fraction showed a single peak in HPLC analysis.

(4) MALDI-TOFMS Analysis The molecular ion peaks of the obtained fractions were measured according to the "MALDI-TOFMS analysis method." The results are shown in Table 37. The m/z value of the most intense peak was considered to be the molecular ion peak of [M+Na ⁺ ] or [M+K ⁺ ]. Furthermore, since the arabinose content in the isolated polysaccharide was 6% or less, it was estimated that the polysaccharide was composed of only galactose or contained one or two molecules of arabinose per molecule. Therefore, the number of galactose and arabinose molecules per molecule and the degree of polymerization (DP) were estimated based on the molecular ion peak m/z value.

The estimated degree of polymerization for each fraction is shown in Table 37. The sugars contained in the major peak fraction were mainly composed of galactose, and the degree of polymerization (DP) matched the peak number. The sugars contained in the minor peak fraction were mainly polysaccharides containing one molecule of arabinose with the remainder being galactose, and the degree of polymerization (DP) matched the peak number plus 0.5.

(5) Evaluation of Degree of Polymerization, Catalytic Activity, and Stereoselectivity The degree of polymerization (DP), catalytic activity, and stereoselectivity of the polysaccharides contained in each fraction obtained by the preparative separation shown in Tables 35 to 37 are shown in Figure 9.

Catalytic activity tended to be better with a higher degree of polymerization, and stereoselectivity tended to be better with a higher degree of polymerization between DP11 and DP15, but was comparable above DP15. From the perspective of stereoselectivity, a degree of polymerization of 13 or higher was considered to be more practical.

(6) ODS-HPLC Separation The fraction "NG19.5mer" obtained above was dissolved in purified water to a concentration of 10 mg/mL, centrifuged, and filtered through a 0.2 μm filter. The resulting filtrate was separated by ODS-HPLC under the following conditions. The corresponding fractions were mixed, the MeOH was removed using an evaporator, and the mixture was freeze-dried.

<ODS-HPLS preparative conditions>
Column: Open column equipped with a guard column (Mightysil RP-18GPAqua, 5 μm, 10 mm I.D. × 250 mm)
Eluent: methanol/purified water (0.5/99.5)
Injection volume: 100μL (=1mg)
Flow rate: 4 mL/min Column temperature: 40°C
Fraction volume: 4 mL
Detector: RID

The chromatogram of the ODS-HPLC fractionation is shown in Figure 10. The peaks enclosed by dashed lines were collected and named 19.5-1 to 19.5-8 in the order of elution. The collected fractions were each concentrated using an evaporator and freeze-dried to obtain samples "19.5-1 to 19.5-8." When each sample was analyzed using the ODS analysis conditions shown below, it was confirmed that 19.5-1, 19.5-2, and 19.5-3 were single peaks. Figure 11 is a graph showing the chromatograms of each sample overlaid.

<ODS analysis>
Column: Open column (Mightysil RP-18GP Aqua, 5 μm, 4.6 mm I.D. × 250 mm)
Eluent: methanol/purified water (1.0/99.0)
Sample concentration: 1 mg/mL
Injection volume: 10μL
Flow rate: 1.0 mL/min Column temperature: 40°C
Detector: RID

The catalytic activity, stereoselectivity, and molecular ion peak of each fraction obtained are shown in Table 38. Catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method IV," and the molecular ion peak was measured according to "MALDI-TOFMS Analysis." The 19.5mer contained a polysaccharide (19.5-3) consisting of 19 galactose molecules and one arabinose molecule, which showed a single peak in HPLC.

Example A11: ODS-HPLC fractionation Fraction BG2 (20 mg) obtained in Example A7 above was dissolved in 400 μL of purified water, and ODS-HPLC fractionation was carried out under the following conditions.

<ODS-HPLC preparative conditions>
Column: YMC-Pack ODS-AQ (6 mm ID x 300 mm (S-5 μm, 12 nm, manufactured by YMC Co., Ltd.)
Eluent: 1.5% methanol Flow rate: 1.7 mL/min Column temperature: 37°C
Detector: RID
Fractionation device: LC-Forte/R (manufactured by YMC Co., Ltd.)

The preparative chromatogram is shown in Figure 12. As shown in Figure 12, fractions corresponding to peaks A to I were collected, and this process was repeated several times. Each was then freeze-dried to obtain 5 to 13 mg of powder. Figure 13 shows the chromatograms obtained by ODS-HPLC analysis of each fraction before and after preparative analysis. The analytical conditions are as follows. Fractions C to I corresponding to peaks C to I all had single peaks.

<Analysis conditions>
Column: YMC-Pack ODS-AQ (250 mm x 4.6 mm ID (S-5 μm, 12 nm, manufactured by YMC Co., Ltd.)
Eluent: 1.5% methanol Column temperature: 37°C
Flow rate: 1.0 mL/min. Detector: RID (device name: Shodex RI-101, manufactured by Showa Denko K.K.)

Fractions D, E, and F were analyzed for monosaccharide composition according to the "monosaccharide composition analysis method," and molecular ion peak measurement was performed according to the "MALDI-TOFMS analysis method." The results are shown in Table 39. The analysis results indicated that fractions D, E, and F are polysaccharides composed of only 20, 21, and 22 galactose units, respectively.

Similarly, fraction BG3 obtained in Example A7 above was separated by ODS-HPLC to obtain fractions corresponding to DP16 to DP19.

As described above, DP16 to DP25 were obtained, and the catalytic activity and stereoselectivity of each fraction were measured using "Catalytic Activity Measurement Method III." The results are shown in Figure 14. Figure 14(a) is a graph showing the catalytic activity of DP16 to DP25, and Figure 14(b) is a graph showing the stereoselectivity of DP16 to DP25. In the range of DP20 to DP25, catalytic activity was high, and there was a tendency for catalytic activity to increase with increasing degree of polymerization. Furthermore, in the range of DP16 to DP20, there was a tendency for stereoselectivity to increase with increasing degree of polymerization, and DP20 to DP25 showed similar stereoselectivity.

Example A12: Modification of reducing ends of low-molecular-weight polysaccharide fractions (1) Conversion of fractions BG1 to BG5 with ethyl 4-aminobenzoate (ABEE) and tert-butyl 4-aminobenzoate (ABtB) Sodium cyanoborohydride (35 mg) and ABEE (165 mg) or ABtB (193 mg) were placed in a screw tube, and methanol (350 μL) and acetic acid (41 μL) were added. The mixture was stirred with a vortex mixer and heated to about 60°C to dissolve, thereby preparing a derivatization reagent.

Each solution of BG1 to BG5 (10 mg/mL, 10 μL) obtained in Example A6 was placed in a 1.5 mL minitube, and derivatization reagent (40 μL) was added. The mixture was then heated at 80°C for 1 hour. Purified water (500 μL) was added, followed by extraction with chloroform. The aqueous layer was analyzed by HPLC under the following conditions.

<HPLC analysis>
Column: TSKGel ODS120H (Tosoh Corporation)
Eluent: sodium borate buffer (pH 9.0)/acetonitrile (91/9)
Flow rate: 1.0 mL/min Column temperature: 35°C
Detector: UV (detection wavelength: 310 nm)

HPLC analysis showed that ABEE-modified BG1-3 each showed a broad single peak, while ABEE-modified BG4-5 each showed multiple peaks. Furthermore, ABtB-modified BG1 showed a broad single peak, while ABtB-modified BG2-3 showed sawtooth peaks. It was found that all of BG1-5 were converted to derivatives with absorption at a wavelength of 310 nm.

(2) Derivatization of SFD2/3 degD1 from water-soluble soybean polysaccharides (ABEE)
A 100 mL round-bottom flask was charged with ABEE (660 mg), sodium cyanoborohydride (140 mg), methanol (20 mL), and acetic acid (163 μL). 20 mg/mL SFD2/3 degD1 (5 mL) obtained in Example A9 was added to the flask, and the mixture was heated at 80°C for 18 hours with stirring. Excess ABEE was removed with chloroform, and the mixture was neutralized with saturated aqueous sodium bicarbonate. Ethanol (four times the volume of the aqueous layer after neutralization) was added to the neutralized solution. The precipitate was collected, dialyzed, and then lyophilized to obtain 29.5 mg of powder (NG-ABEE).

NG-ABEE and the unmodified sample (NG) were analyzed using the "Gel Filtration HPLC Analysis II" method. The results are shown in Figure 15. Detection was performed using both RID and UV (detection wavelength: 310 nm). NG-ABEE was confirmed to have absorption at a wavelength of 310 nm using UV detection, and RID detection revealed that the retention times of NG and NG-ABEE were almost the same.

The catalytic activity and stereoselectivity of NG and NG-ABEE were evaluated. Catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method III." The results are shown in Table 40. NG-ABEE exhibited the same stereoselectivity as NG and 1.3 times higher catalytic activity.

(3) ABEE Conversion and Catalytic Activity of Low Molecular Weight Polysaccharide Fraction The fraction AM-LLMW obtained in Example A7 was converted to ABEE in the same manner.

When the aqueous layer was analyzed using the "Gel Filtration HPLC Analysis II" method, a peak with absorption at a wavelength of 310 nm was detected. This confirmed that fraction AM-LLMW could also be converted to ABEE.

Example B1: NMR analysis ¹ H-NMR and ¹³ C-NMR of AL-LMW were measured under the following conditions.
Solvent: Heavy water ( _D2O )
NMR measurement device: Bruker AVANCE II 400 (manufactured by Bruker)
Frequency: 400 MHz for ¹ H-NMR, 100 MHz for ¹³ C-NMR

^{The 1} H-NMR spectrum is shown in Figure 16, and ^{the 13} C-NMR spectrum is shown in Figure 17. The chemical shift values (δ) of each were analyzed with reference to the literature (Food Chemistry, 242, 211-216 (2018)). As a result, the NMR spectrum of AL-LMW was similar to the values derived from (→4)-β-D-Galp-(1→) described in the literature, indicating that the main structure of AL-LMW is a galactan in which galactose is β1,4-linked. Furthermore, no peaks corresponding to olefins or carbonyl groups were observed in the NMR spectrum.

Example B2: Multi-step derivatization of fractions NG3-4 kDa and analysis of sugar chain binding positions [0112] Using the 11-mer, 11.5-mer, 15-mer, 15.5-mer, 19.5-mer, or 20-mer prepared from the fractions NG3-4 kDa obtained in Example A10 as samples, multi-step derivatization (methylation, monosaccharification, alditolization, and acetylation) was performed, and the sugar chain binding positions were analyzed based on the difference in molecular weight.
(Step 1) Methylation A sample (1 mg) was weighed into a 2 mL screw-cap tube, and suspended in dimethyl sulfoxide (500 μL) and sodium hydroxide (50 mg) ground into powder in a mortar, followed by stirring at 1000 rpm for 30 minutes at 25° C. Methyl iodide (250 μL) was added, mixed, and then stirred at 25° C. for 20 to 25 hours.
(Step 2) Monosaccharification: 100 mM aqueous sodium thiosulfate solution (300 μL) and dichloromethane (300 μL) were added to the methylated composition, and the organic layer was recovered. The organic layer was washed with 100 mM aqueous sodium thiosulfate solution, desalted with purified water, and the organic solvent was removed using a centrifugal evaporator. The resulting intermediate mixture was suspended in purified water (150 μL), and 4 M trifluoroacetic acid (final concentration: 2 M) (150 μL) was added and stirred, followed by shaking at 120°C for 2 hours.
(Step 3) Alditol Conversion The composition after monosaccharification was cooled to room temperature and then freeze-dried. Sodium borodeuteride (20 mg) and 2 M aqueous ammonia (500 μL) were added and dissolved, and the mixture was shaken at 60° C. for 2 hours.
(Step 4) Acetylation After the alditol-containing composition was cooled to room temperature, methanol (500 μL) was added, and the solvent was distilled off by repeated azeotropic distillation. Acetic anhydride (400 μL) and trifluoroacetic acid (100 μL) were added to the resulting residue, and the mixture was shaken at 60°C for 2 hours. After the resulting composition was cooled to room temperature, saturated sodium bicarbonate (400 μL) and chloroform (400 μL) were added, and the organic layer was recovered. The organic layer was neutralized with saturated sodium bicarbonate, washed with purified water, and dried over anhydrous sodium sulfate to obtain an acetylated sample. The acetylated sample was analyzed by GC-MS.

<GC-MS conditions>
Measuring equipment: GC2010/GCMS-QP2010 (Shimadzu Corporation)
Analytical column: DB-225MS (20 m x 0.25 mm ID, 0.25 μm)
Carrier gas: helium Column temperature: After maintaining 140°C for 1 minute, the temperature was increased to 220°C at a rate of 20°C/min and maintained at 220°C for 25 minutes.

Figure 18 shows the GC spectra detected at m/z 118 after derivatization of the 19.5mer and 20mer. The peak detected at m/z 118 is a peak that commonly appears in mass spectra, indicating that some of the hydrogen atoms in partially methylated alditol acetates derived from hexoses have been replaced with deuterium (see J. Biol. Chem., 293(30), 11955-11965(2018)). Furthermore, according to the spectral library described below, many of the peaks derived from arabinose are detected at m/z 118.

GC-MS analysis revealed three peaks at retention times of 9.450 minutes (a), 9.583 minutes (b), and 12.517 minutes (c) detected at m/z 118. The mass spectra of each peak were compared with data from the spectral library provided by the Complex Carbohydrate Research Center at the University of Georgia (https://glygen.ccrc.uga.edu/ccrc/specdb/ms/pmaa/pframe.html). The peaks were identified as corresponding to (a) 1,4-arabinopyranose or 1,5-arabinofuranose, (b) terminal galactopyranose, and (c) 1,4-galactopyranose, in order of decreasing retention time. These results suggest that the 19.5-mer and 20-mer (neutral galactans) are unbranched glycans composed primarily of 1,4-linked galactose, with arabinose inserted between the glycans.

Example C: Investigation of allergen content (1) Analysis of allergen content Solutions or suspensions of okara (trade name: Okara Powder, manufactured by Acrosia Co., Ltd.), water-soluble soy polysaccharides (trade name: SOYAFIVE S-DN), polysaccharide fraction AL-LMW, and low molecular weight polysaccharide fraction AM-LLMW were prepared, and the soy allergen content in the supernatant was measured using "FASTKIT Slim Soy" (manufactured by Nippon Meat Packers Co., Ltd.) according to the instruction manual. Purified water was added to the okara to extract, followed by centrifugation, and the resulting supernatant was subjected to analysis.

At the sample concentration (normal concentration) prepared according to the instructions, allergens in the sample exceeding 5 μg/g are detected as positive. No allergens were detected in the water-soluble soy polysaccharides, but when the sample concentration was increased five-fold and re-measured, allergens were detected. AL-LMW and AM-LLMW were measured at a five-fold increased sample concentration. The results are shown in Table 41. The polysaccharide fraction had lower soy allergen levels than the water-soluble soy polysaccharides, and the soy allergen levels were found to be reduced to undetectable levels in the low molecular weight polysaccharide fraction.
++: Positive bands appear dark.
+: A faint positive band is visible.
-: No positive bands observed.

Example C2: Resistance to gelation of allergens 20 mg of each of water-soluble soybean polysaccharide (SOYAFIVE S-DN, Fuji Oil Co., Ltd.), two polysaccharide fractions (LP-LMW, AL-LMW-EtOH 0.8/1.1 PPT), and a low molecular weight polysaccharide fraction (AM-LLMW-EtOH 1.0/1.4) were placed in a 1.5 mL glass container and suspended or dissolved in saturated saline to make a 100 μL solution. After leaving this at room temperature for 1 day, the container was inverted. If the solution did not flow, it was judged to have gelled.
The water-soluble soybean polysaccharides gelled, but the polysaccharide fractions (LP-LMW, AL-LMW-EtOH 0.8/1.1 PPT) and the low molecular weight polysaccharide fraction (AM-LLMW-EtOH 1.0/1.4) did not gel.

Example C3: Comparison of Stereoselectivity Stereoselectivity was evaluated (n=5) using a polysaccharide sample (7 mg) according to "Catalytic Activity Measurement Method III." The polysaccharide samples used were water-soluble soybean polysaccharide (SOYAFIBE S-DN, manufactured by Fuji Oil Co., Ltd., molecular weight approximately 100,000 to 840,000), LP-LMW (molecular weight 30,000 to 50,000), AL-LMW (molecular weight 30,000 to 50,000), or AM-LLMW (molecular weight 3000 to 5000). The same experiment was also performed using saturated potassium chloride aqueous solution instead of saturated saline. Significant differences were determined by a t-test against water-soluble soybean polysaccharides, and a p<0.01 result was considered to be "significantly different."

The results are shown in Table 42. As the molecular weight of the water-soluble soybean polysaccharide was reduced, the stereoselectivity improved. Furthermore, it was found that the use of saturated potassium chloride aqueous solution rather than saturated saline solution in the epoxy ring-opening reaction resulted in higher stereoselectivity.

Example C4: Improvement of product recovery yield by two-phase reaction 20 mg of SOYAFIBE S-DN (Fuji Oil Co., Ltd.) or low molecular weight polysaccharide fraction (AM-LLMW) and 300 μL of saturated saline were placed in a 1.5 mL glass container, and 300 μL of a toluene solution of the substrate (containing 10 v/v % epoxycyclohexane and 10.8 v/v % cyclopropylamine) was added, followed by stirring at 300 rpm for 22 hours at 37°C. The toluene layer was then collected, and a fresh toluene solution of the substrate (300 μL) was added. This process was repeated two more times. In other words, the aqueous phase was reused, and a total of three reactions were carried out.

The content of the product (2-cyclopropylaminocyclohexanol) in the recovered toluene layer was measured by GC analysis according to "Catalytic Activity Measurement Method I".

The results are shown in Table 43. Repeated use of both the water-soluble soybean polysaccharide and AM-LLMW did not change the conversion rate, the amount of product recovered, or the optical purity. On the other hand, repeated use of the water-soluble soybean polysaccharide caused a portion of the reaction solution to gel and adhere to the inner wall of the vessel, making recovery of the toluene layer difficult and resulting in a low amount of product recovered. Figure 19 is a photograph showing the state of each reaction vessel before the toluene layer was recovered. The left photograph shows the reaction vessel using water-soluble soybean polysaccharide, and the right photograph shows the reaction vessel using AM-LLMW. In Figure 19, the solid arrow indicates the liquid level of the toluene layer, and the dashed arrow indicates the interface between the water layer and the toluene layer.

Example C5: High-Temperature Reactivity Using SOYAFIBE S-DN or LP-LMW, catalytic activity and stereoselectivity were measured by Activity Measurement Method V at reaction temperatures of 40°C, 50°C, 60°C, and 70°C. The results are shown in Table 44. Both LP-LMW and water-soluble soybean polysaccharides reached their maximum at 60°C. Stereoselectivity was hardly affected by temperature. At all temperatures, LP-LMW maintained a specific activity 1.5 to 2 times higher than SOYAFIBE S-DN.

Example D: Preparation of Amino Alcohol The racemic amino alcohol used as a standard for the synthesized product was analyzed by GC and HPLC under the following conditions. The analytical conditions are shown in Tables 45 and 46.

<GC analysis conditions>
Column size: (30m x 0.25mm x 0.25μm)

<Chiral HPLC analysis conditions>
Detection wavelength: 254 nm
Column used: CHIRALPAK ID (4.6 mm x 250 mm, 5 μm, manufactured by DAICEL)
Injection volume: 10μL

The synthesis of the racemate for analysis and the analytical method are described in the following Reference Examples.
Reference Example 1 Synthesis of trans-2-((4-methoxybenzyl)amino)cyclohexanol 0.98 g of 1,2-epoxycyclohexane and 1.37 g of 4-methoxybenzylamine were weighed into a 50 mL recovery flask, and 5.3 mL of methanol, 1.3 mL of water, and 85 mg of lithium chloride were added, followed by stirring at 50°C for 48 hours. The reaction solution was extracted three times with ethyl ether, and the organic layer was washed successively with purified water and saturated brine, dried over anhydrous sodium sulfate, and concentrated. The product was purified by silica gel column chromatography (chloroform:methanol:triethylamine=480:12.2:1, gel 200 mL) to obtain 2.18 g of the title compound as a white powder (yield 92.9%).
1H-NMR(CDCl3, 400MHz) δ 0.97(1H, m), 1.25(3H, m), 1.72(2H, m), 2.03(1H, m), 2.17(1H, m),2.27(1H, m), 3.18(1H, dt, J=4.4, 9.8Hz), 3.63(1H, d, J=12.7Hz), 3.79(3H, s),3.88(1H, d, J=12.7Hz), 6.86(2H, d, J=8.7Hz), 7.23(2H, d, J=8.7Hz)
13C-NMR(CDCl3, 100MHz) δ 24.3, 25.2, 30.6, 33.2, 50.1, 55.3, 63.0, 73.9, 113.8, 129.2, 132.8
Analysis condition A 22.9 minutes [epoxide: 4.1 minutes, amine: 13.3 minutes]
Analysis conditions α 42.1 minutes: (1S, 2S) isomer, 45.6 minutes: (1R, 2R) isomer

Reference Example 2 Synthesis of trans-4-((4-methoxybenzyl)amino)tetrahydrofuran-3-ol In a 10 mL screw-cap test tube, 137 mg of 4-methoxybenzylamine was dissolved in 533 μL of methanol and 133 μL of purified water, and 86 mg of 3,4-epoxytetrahydrofuran and 8.5 mg of lithium chloride were added sequentially, followed by vigorously stirring in an oil bath at 50° C. for 48 hours. The reaction solution was extracted three times with ethyl ether, and the organic layer was washed sequentially with purified water and saturated brine, dried over anhydrous sodium sulfate, and concentrated. The product was purified by silica gel column chromatography (chloroform:methanol:triethylamine=50:1:1, gel 25 mL) to obtain 184.6 mg of the title compound as a yellow powder (yield 82.8%).
1H-NMR(CDCl3, 400MHz) δ 1.89(2H, br), 3.20(1H, m), 3.55(1H, m), 3.68(1H, m), 3.74(2H, s),3.79(3H, s), 3.98(1H, m), 4.06(1H, m), 4.16(1H, m), 6.86(2H, m), 7.22(2H, m)
13C-NMR(CDCl3, 100MHz) δ 51.7, 55.3, 66.1, 72.5, 74.1, 76.8, 113.9, 129.4, 131.8, 158.8
Analysis condition B 28.3 minutes [epoxide: 6.2 minutes, amine: 8.8 minutes]
Analysis conditions χ 38.2 minutes: (3S,4R) isomer, 41.1 minutes: (3R,4S) isomer [Amine: 21.2 minutes]

Reference Example 3 Synthesis of trans-2,2-dimethyl-6-(benzylamino)-1,3-dioxepan-5-ol Using 144 mg of 4,4-dimethyl-3,5,8-trioxabicyclo[5.1.0]octane and 107 mg of benzylamine, a reaction was carried out in the same manner as in Reference Example 2, and the reaction product was purified by silica gel column chromatography (ethyl acetate:hexane:triethylamine=50:1:1, gel 30 mL) to give 210.0 mg of the title compound as a brown powder (yield 83.7%).
1H-NMR(CDCl3, 400MHz) δ 1.33(3H, s), 1.34(3H, s), 2.56(1H, m), 3.54(3H, m), 3.80(3H, m),3.93(1H, d, J=13.2Hz), 7.27(5H, m)
13C-NMR(CDCl3, 100MHz) δ 24.6, 24.8, 51.4, 59.2, 61.5, 61.8, 72.1, 101.4, 127.1, 128.1,128.5, 140.4
Analysis condition A 21.5 minutes [epoxide: 10.1 minutes, amine: 8.4 minutes]
Analysis conditions δ 22.0 min: (5S,6R) isomer, 28.3 min: (5R,6S) isomer [amine: 13.5 min]

Reference Example 4: Synthesis of trans-2,2-dimethyl-6-((4-methoxybenzylamino)-1,3-dioxepan-5-ol Using 144 mg of 4,4-dimethyl-3,5,8-trioxabicyclo[5.1.0]octane and 137 mg of 4-methoxybenzylamine, 238.9 mg of the title compound was obtained as a white powder (yield 85.0%) in the same manner as in Reference Example 2.
1H-NMR(CDCl3, 400MHz) δ 1.33(3H, s), 1.34(3H, s), 2.55(1H, m), 3.52(3H, m), 3.73(1H, d,J=12.9Hz), 3.77(1H, m), 3.80(4H, m including singlet), 3.85(1H, d, J=12.9Hz), 6.86(2H, m), 7.25(5H, m)
13C-NMR(CDCl3, 100MHz) δ 24.6, 24.8, 50.8, 55.3, 59.2, 61.4, 61.9, 72.1, 101.6, 113.9, 129.3, 132.5, 158.5
Analysis condition A 24.1 minutes [epoxide: 10.1 minutes, amine: 13.3 minutes]
Analysis conditions ε 25.4 minutes: (5S,6R) isomer, 27.4 minutes: (5R,6S) isomer

Reference Example 5 Synthesis of trans-2,2-dimethyl-6-(2-propynylamino)-1,3-dioxepan-5-ol [0123] The procedure was repeated in the same manner as in Reference Example 2, except that 144 mg of 4,4-dimethyl-3,5,8-trioxabicyclo[5.1.0]octane and 112 mg of propargylamine were used and purification was performed by silica gel column chromatography (chloroform:methanol:triethylamine=100:3:3, gel 20 mL), to obtain 144.8 mg of the title compound as a brown powder (yield 85.7%).
1H-NMR(CDCl3, 400MHz) δ 1.33(3H, s), 1.35(3H, s), 1.63(1H, br), 2.23(1H, t, J=2.5Hz),2.60(1H, brd, J=6.9Hz), 2.70(1H, m), 3.51(3H, m), 3.55(1H, m), 3.59(1H, m),3.81(1H, m), 3.83(1H, m)
13C-NMR(CDCl3, 100MHz) δ 24.6, 24.7, 36.5, 59.2, 61.3, 61.7, 71.5, 71.9, 82.4, 101.5
Analysis condition G 32.0 minutes, 32.4 minutes [epoxide: 5.5 minutes]

Reference Example 6 Synthesis of trans-2,2-dimethyl-6-(cyclopropylamino)-1,3-dioxepan-5-ol [0123] The procedure was repeated in the same manner as in Reference Example 2, except that 177.6 mg of 4,4-dimethyl-3,5,8-trioxabicyclo[5.1.0]octane and 570 mg of cyclopropylamine were used and purification was performed by silica gel column chromatography (ethyl acetate:hexane:triethylamine=100:1:1, gel 25 mL), to obtain 226.0 mg of the title compound as a white powder (yield 91.2%).
1H-NMR(CDCl3, 400MHz) δ 0.29(1H, m), 0.43(3H, m), 1.33(3H, s), 1.35(3H, s), 2.24(1H, m),2.35(2H, br), 2.66(1H, m), 3.46(1H, m), 3.54(2H, m), 3.74(1H, m), 3.84(1H, m)
13C-NMR(CDCl3, 100MHz) δ 6.8, 7.2, 24.6, 24.8, 28.2, 59.8, 61.8, 62.6, 71.9, 101.3
Analysis condition G 32.0 minutes, 32.4 minutes [epoxide: 5.5 minutes]

Reference Example 7: Synthesis of (1R,2R)-2-(benzylamino)cyclohexan-1-ol In a 25 mL recovery flask, 35.4 mg of benzaldehyde was dissolved in 7 mL of methanol, and with vigorously stirring, 50.6 mg of (1R,2R)-2-aminocyclohexanol hydrochloride, 38 μL of acetic acid, and 78.6 mg of sodium cyanoborohydride were added sequentially, followed by vigorously stirring for 19 hours. The reaction solution was concentrated and extracted three times with chloroform. The combined organic layer was washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated. The concentrate was purified by PTLC (n-butanol:acetic acid:water=4:1:1) to give 19.2 mg of the title compound as a white powder (yield 25.6%).
Analysis conditions α 22.2 minutes (>99%ee)

Reference Example 8: Synthesis of (1R,2R)-2-((4-methoxybenzyl)amino)cyclohexan-1-ol A reaction was carried out in the same manner as in Reference Example 7 using 45.4 mg of 4-methoxybenzaldehyde and 50.6 mg of (1R,2R)-2-aminocyclohexanol hydrochloride, and the reaction was purified by PTLC (n-butanol:acetic acid:water=4:1:1) to obtain 10.5 mg of the title compound as a white powder (yield 52.5%).
Analysis conditions β 45.7 minutes (>99%ee)

Reference Example 9: Synthesis of (3S,4R)-4-(benzylamino)tetrahydrofuran-3-ol. In a 10 mL recovery flask, 23.3 mg of benzaldehyde was dissolved in 6 mL of methanol. With vigorous stirring, 22.9 mg of (3S,4R)-4-amino-tetrahydrofuran-3-ol, 19.2 μL of acetic acid, and 45.5 mg of sodium cyanoborohydride were added sequentially, and the mixture was stirred vigorously for 69 hours. The reaction solution was concentrated and extracted three times with chloroform. The organic phase was washed successively with water and saturated brine, dried over anhydrous sodium sulfate, and concentrated. The concentrate was purified by PTLC (chloroform:methanol:triethylamine=20:1:1) to give 3.1 mg of the title compound as a brown oil (yield 10.0%).
Analysis conditions β 27.6 minutes (>99%ee)

Reference Example 10: Synthesis of (3S,4R)-4-((4-methoxybenzyl)amino)tetrahydrofuran-3-ol Using 27.2 mg of 4-methoxybenzaldehyde and 20.6 mg of (3S,4R)-4-amino-tetrahydrofuran-3-ol, a reaction was carried out in the same manner as in Reference Example 9. The product was purified by PTLC (CHCl ₃ :MeOH:Et ₃ N=20:1:1) to give 5.9 mg of the title compound as a yellow powder (yield 16.5%).
Analysis conditions χ 36.6 minutes (>99%ee)

Reference Example 11: Synthesis of (5R,6S)-2,2-dimethyl-6-(benzylamino)-1,3-dioxepan-5-ol Under a nitrogen atmosphere, 5.7 mg of (S)-BINOL was dissolved in 0.5 mL of anhydrous toluene in a 10 mL recovery flask, and 20 μL of a 0.5 M solution of titanium tetraisopropoxide in anhydrous toluene was added under vigorously stirring. After stirring for 10 minutes, 117 mg of benzylamine, 2.7 μL of purified water, and 144 mg of 4,4-dimethyl-3,5,8-trioxabicyclo[5.1.0]octane were added sequentially, and the mixture was vigorously stirred for 18.5 hours. 2 mL of ethyl acetate was added to the reaction solution, which was then suction filtered using Radiolite #700 as a filter aid and concentrated. The concentrate was purified by silica gel column chromatography (petroleum ether:ethyl acetate:triethylamine=40:20:3, gel 30 mL) to give 128.8 mg of the title compound as a white powder (yield 51.3%).
Analysis conditions δ 28.3 minutes (95.3% ee)

Reference Example 12: Synthesis of (5R,6S)-2,2-dimethyl-6-((4-methoxybenzyl)amino)-1,3-dioxepan-5-ol Using 144 mg of 4,4-dimethyl-3,5,8-trioxabicyclo[5.1.0]octane and 151 mg of 4-methoxybenzylamine, reaction and extraction were carried out in the same manner as in Reference Example 11, and the resulting mixture was purified by silica gel column chromatography (petroleum ether:ethyl acetate:triethylamine=40:20:3, gel 30 mL) to obtain 91.6 mg of the title compound as a white powder (yield 32.6%).
Analysis conditions δ 26.6 min (69.7% ee)

<Reaction of producing various amino alcohols using polysaccharide fractions>
10 mg of SOYAFIBE S-DN or 5 mg of LP-LMW or AL-LMW was weighed into an HPLC vial, and 100 μL of a toluene solution containing 1 M epoxide and 1.5 M amine was added. 6 μL of saturated saline was added, and the reaction was carried out at 37±1°C with vigorous stirring. At 1, 3, 6, 24, and 48 hours, 4 μL of the reaction mixture was sampled, diluted 50-fold with toluene, dehydrated with anhydrous sodium sulfate, filtered with cotton, and then subjected to GC analysis. Activity values were calculated using the effective carbon number method from the areas of the starting epoxide and the product amino alcohol. Stereoselectivity (optical purity of the product amino alcohol) was calculated by chiral GC (conditions C-G) or chiral HPLC (conditions α-ε). The polysaccharide fraction showed catalytic activity 1.1 to 2.5 times higher than that of water-soluble soybean polysaccharides.

<Catalytic activity of water-soluble soybean polysaccharides and LMW toward various substrates>
The results are shown in Tables 47 to 49.
Bn: benzyl group, PMB: p-methoxybenzyl group, Not tested: not tested
Bn: benzyl group, PMB: p-methoxybenzyl group, Not tested: not tested
Bn: benzyl group, PMB: p-methoxybenzyl group, Not tested: not tested

<Reaction of producing various amino alcohols using polysaccharide fractions 2>
10 mg of AL-LMW (30 mg for the reaction of 3,4-epoxyhexane and cyclopropylamine) was weighed into a 2 mL glass bottle, 200 μL of a toluene solution containing 1 M epoxide and 1.5 M amine was added, and 12 μL of a saturated aqueous sodium chloride solution or a saturated aqueous potassium chloride solution was added. The reaction was carried out at 37±1°C with vigorously stirring for 3 days. The reaction solution was sampled and subjected to chiral GC analysis (conditions H to L in Table 45). The conversion rate was calculated using the effective carbon number method from the areas of the starting epoxide and the product amino alcohol, and the stereoselectivity was calculated from the enantiomeric excess of the product amino alcohol.

The results are shown in Table 50. A 2 mL glass bottle was charged with 800 μL of ethanol containing 2 M epoxide and 2 M amine, and the mixture was stirred at 1000 rpm at 37° C. for 48 hours to prepare a racemic β-amino alcohol, which was then analyzed to confirm the retention times of the enantiomers of the product.

<Reaction of various amino alcohols produced by polysaccharide fractions 3>
10 mg of AL-LMW was weighed into a 2 mL glass bottle, and 200 μL of a toluene solution containing 1 M epoxycyclohexane and either 1.5 M 2-amino-3-methylbutane or sec-butylamine was added. 12 μL of saturated saline was added, and the reaction was carried out at 37 ± 1°C with vigorously stirring for 24 hours. The reaction solution was sampled and subjected to chiral GC analysis (H or J in Table 45). The conversion rate was calculated using the effective carbon number method from the sum of the isomer areas of the starting epoxide and the resulting amino alcohol, and the isomer ratio was calculated from the area of the amino alcohol isomer. The results are shown in Table 51. When either of the two chiral amines was used as the substrate, the polysaccharide selectively produced one of the four isomers that could be produced by the reaction with the epoxide at a ratio of 60% or more.

Example D2: Reaction for Producing Aminoalcohol from 2-Chlorocyclohexanol and Cyclopropylamine 40 mg of AM-LLMW was weighed into a 2 mL glass bottle, and 800 μL of saturated saline containing 11 mM 2-chlorocyclohexanol and 500 mM cyclopropylamine was added. The mixture was stirred at 37±1°C. The reaction solution was sampled 20 minutes and 1 hour after the start of stirring, extracted with toluene, and subjected to chiral GC analysis (Table 45, H). The concentrations of each component in the reaction solution were calculated and are shown in Table 52. Even without the addition of polysaccharide, 2-chlorocyclohexanol was partially converted via 1,2-epoxycyclohexane to nearly racemic 2-cyclopropylaminocyclohexanol; however, with the addition of polysaccharide, it was stereoselectively converted to 2-cyclopropylaminocyclohexanol.

Example E: Galactan without catalytic activity Catalytic activity was measured for commercially available polysaccharides containing arabinogalactans. The catalytic activity was measured according to "Catalytic Activity Measurement Method I" or "Catalytic Activity Measurement Method III."

The results are shown in Table 53. The commercially available polysaccharides containing arabinogalactans had significantly lower catalytic activity or stereoselectivity than the polysaccharides of the present invention.

Example F1: Preparation of Gal Donor Galactose donor (S6) was prepared as follows.
The abbreviations used should be understood as having their meaning well known in the art of organic chemistry, for example:
Bn: benzyl PhCH(OMe) ₂ : benzaldehyde dimethyl acetal TsOH: para-toluenesulfonic acid DMF: N,N-dimethylformamide MP: 4-methoxyphenyl TFA: trifluoroacetic acid Tol: para-toluoyl CACl: trichloroacetyl chloride CA: chloroacetyl pyr: pyridine Mbp: 5-tert-butyl-2-methylphenyl DMTST: dimethyl(methylthio)sulfonium triflate DTBMP: 2,6-di-tert-butyl-4-methylpyridine HSMbp: 5-tert-butyl-2-methylbenzenethiol DABCO: 1,4-diazabicyclo[2.2.2]octane CAN: ammonium cerium(IV) nitrate
DBU: 1,8-diazabicyclo[5.4.0]-7-undecene TMSOTf: trimethylsilyl triflate AcOEt: ethyl acetate MS4A: molecular sieves 4A
THF: tetrahydrofuran

[1] Synthesis of 4-methoxyphenyl 3-O-benzyl-4,6-O-benzylidene-β-D-galactopyranoside (Compound S2) 4-Methoxyphenyl 3-O-benzyl-β-D-galactopyranoside (Compound S1, Tokyo Chemical Industry Co., Ltd., 200 g, 0.53 mol) was dissolved in N,N-dimethylformamide (1 L), and benzaldehyde dimethyl acetal (120 mL, 1.5 equivalents) and p-toluenesulfonic acid monohydrate (10 g, 0.1 equivalents) were added, followed by stirring at room temperature for 1 day. The reaction solution was neutralized with triethylamine (7.4 mL), and the precipitated solid was filtered off. The filtrate was concentrated under reduced pressure. The resulting residue was recrystallized from ethyl acetate-isopropyl ether to give Compound S2 (237 g, 96% yield).
¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 2.52 (d, 1 H, J = 2.3 Hz, OH), 3.46 (s, 1 H, H-5), 3.57 (dd, 1 H, J = 3.7 Hz, 9.6 Hz, H-3), 3.77 (s, 3 H, PhOMe), 4.05 (dd, 1 H, J = 1.9 Hz, 12.4 Hz, H-6), 4.19 (d, 1 H, J = 2.8 Hz, H-4), 4.24 (m, 1 H, H-2), 4.34 (dd, 1 H, J = 1.2 Hz, 12.4 Hz, H-6'), 4.77, 4.81 (2d, J = 11.9Hz, CH ₂ Ph), 4.70 (d, 1 H, J = 8.2 Hz, H-1), 5.48 (s, 1 H, CHPh), 6.81, 7.07 (2 m, 4 H, PhOMe), 7.30-7.55 (m, 10 H, 2 Ph).

[2] Synthesis of 4-methoxyphenyl 3-O-benzyl-4,6-O-benzylidene-2-O-p-toluoyl-β-D-galactopyranoside (Compound S3) Compound S2 (237 g, 0.51 mol) was dissolved in pyridine (1.2 L), N,N-dimethylaminopyridine (35 g, 0.56 equivalents) was added, and the mixture was heated to 60°C. p-Toluoyl chloride (200 mL, 3 equivalents) was added dropwise, and the mixture was stirred at 60°C for 3 hours, and then cooled to room temperature. Methanol (100 mL) was added to decompose the excess reagent, and the reaction solution was concentrated under reduced pressure. The residue was dissolved in methylene chloride and washed with 2 M hydrochloric acid and saturated aqueous sodium bicarbonate. Magnesium sulfate was added to the organic layer to dry it. The desiccant was removed by filtration, and the solvent was evaporated under reduced pressure. The resulting residue was recrystallized from methylene chloride-isopropyl ether to obtain compound S3 (286 g, yield 96%).
¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 2.44 (s, 3 H, COPhMe), 3.51 (s, 1 H, H-5), 3.72 (s, 3 H, PhOMe), 3.79 (dd, 1H, J = 3.6 Hz, 10.0 Hz, H-3), 4.10 (dd, 1 H, J = 1.4 Hz, 12.4 Hz, H-6), 4.27 (d, 1 H, J = 3.6 Hz, H-4), 4.34 (dd, 1 H, J = 1.4 Hz, 12.4 Hz, H-6'), 4.64, 4.71 (2 d, 2 H, J = 12.8 Hz, CH ₂ Ph), 5.00 (d, 1 H, J = 7.8 Hz, H-1), 5.55 (s, 1 H, CHPh), 5.85 (dd, 1 H, J = 7.8 Hz, 10.0 Hz, H-2), 6.72, 6.93 (2 m, 4 H, PhOMe), 7.20-7.41 (m, 10 H, 2 Ph), 7.60 (m, 2 H, meta- of COPhMe), 7.95 (d, 2 H, ortho- of COPhMe).

[3] Synthesis of 4-methoxyphenyl 3,6-di-O-benzyl-2-O-p-toluoyl-β-D-galactopyranoside (Compound S4) Compound S3 (148 g, 0.25 mol) was dissolved in methylene chloride (1.1 L), triethylsilane (300 mL, 7.5 equivalents) was added, and the mixture was cooled to -40°C. Trifluoroacetic acid (145 mL, 7.5 equivalents) was added dropwise, and the mixture was stirred at -40°C for 1 day. The reaction solution was diluted with methylene chloride and washed with water, saturated aqueous sodium bicarbonate, and saturated brine, in that order. Magnesium sulfate was added to the organic layer to dry it, and the desiccant was filtered off, and the solvent was evaporated under reduced pressure. The resulting residue was recrystallized from methylene chloride-isopropyl ether to give Compound S4 (139 g, yield 93%).
¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 2.43 (s, 3 H, COPhMe), 2.67 (s, 1 H, OH), 3.69 (dd, 1H, J = 3.2 Hz, 9.6 Hz, H-3), 3.71 (s, 3 H, PhOMe), 3.75 (t, 1 H, J = 6.0 Hz, H-5), 3.83 (dd, 1 H, J = 6.4 Hz, 9.7 Hz, H-6), 3.91 (dd, 1 H, J = 5.5 Hz, 9.7 Hz, H-6'), 4.16 (br.s, 1 H, H-4), 4.56, 4.69 (2 d, 2 H, J = 12.4 Hz, CH ₂ Ph), 4.60 (s, 2 H, CH ₂ Ph), 4.91 (d, 1 H, J = 7.8 Hz, H-1), 5.70 (dd, 1 H, J = 7.8 Hz, 9.6 Hz, H-2), 6.71, 6.91 (2 m, 4 H, PhOMe), 7.15-7.37 (m, aromatic), 7.92 (d, 2 H, ortho- of COPhMe).

[4] Synthesis of 5-tert-butyl-2-methylphenyl 3,6-di-O-benzyl-1-thio-2-O-p-toluoyl-β-D-galactopyranoside (Compound S5) Compound S4 (60 g, 103 mmol) was dissolved in methylene chloride (900 mL), and 5-tert-butyl-2-methylbenzenethiol (93 mL, 5 equivalents) and boron trifluoride diethyl ether complex (6.4 mL, 0.5 equivalents) were added. After stirring at room temperature for 15 minutes, the reaction mixture was poured into saturated aqueous sodium bicarbonate (1 L). The organic layer was dried over magnesium sulfate, the desiccant was filtered off, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (elution solvent: hexane:ethyl acetate = 6:1) to obtain Compound S5 (46.1 g, yield 70%).
¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 1.29 (s, 9 H, t-Bu), 2.18 (s, 3 H, PhMe), 2.44 (s, 3 H, COPhMe), 2.59 (s, 1 H, OH), 3.62-3.68 (m, 2 H, H-3, H-5), 3.76 (dd, 1 H, J = 5.5 Hz, 9.6 Hz, H-6), 3.85 (dd, 1 H, J = 6.4 Hz, 9.6 Hz, H-6'), 4.17 (br. s, 1 H, H-4), 4.53, 4.65 (2 d, 2 H, J = 12.4 Hz, CH ₂ Ph), 4.58 (s, 2 H, CH ₂ Ph), 4.67 (d, 1 H, J = 10.0 Hz, H-1), 5.53 (t, 1 H, J = 10.0 Hz, H-2), 7.03-7.58 (m, 15 H, aromatic), 7.92 (d, 2 H, ortho- of COPhMe).

[5] Synthesis of 5-tert-butyl-2-methylphenyl 3,6-di-O-benzyl-4-O-chloroacetyl-1-thio-2-O-p-toluoyl-β-D-galactopyranoside (Compound S6) Compound S5 (46.1 g, 71.9 mmol) was dissolved in methylene chloride (450 mL) and pyridine (90 mL) and cooled to 0°C. A solution of chloroacetyl chloride (23 mL, 4 equivalents) in methylene chloride (120 mL) was added dropwise, and the mixture was stirred at 0°C for 1 hour. The reaction solution was extracted with methylene chloride and washed with 2 M hydrochloric acid and saturated aqueous sodium bicarbonate. The organic layer was dried over magnesium sulfate, the desiccant was filtered off, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (elution solvent: hexane:ethyl acetate=4:1) to obtain Compound S6 (49.4 g, yield 96%).
¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 1.22 (s, 9 H, t-Bu), 2.18 (s, 3 H, PhMe), 2.45 (s, 3 H, COPhMe), 3.54 (dd, 1 H, J = 7.8 Hz, 9.2 Hz, H-6), 3.61 (dd, 1 H, J = 5.5 Hz, 9.2 Hz, H-6'), 3.68 (dd, 1 H, J = 3.2 Hz, 9.6 Hz, H-3), 3.81 (m, 1 H, H-5), 4.04, 4.18 (2 d, 2 H, J = 15.1 Hz, COCH ₂ Cl), 4.44, 4.58, 4.66 (3d, 4 H, J = 12.4 Hz, 2 CH ₂ Ph), 4.67 (d, 1 H, J = 10.0 Hz, H-1), 5.41 (t, 1 H, J = 10.0 Hz, H-2), 5.75 (d, 1 H, J = 3.2, H-4), 6.89-7.50 (m, aromatic), 7.89 (d, 2 H, ortho- of COPhMe).

Example F2: Preparation of Gal(2mer) derivative

[1] Synthesis of 4-methoxyphenyl(3,6-di-O-benzyl-4-O-chloroacetyl-2-O-p-toluoyl-β-D-galactopyranosyl)-(1-4)-3,6-di-O-benzyl-2-O-p-toluoyl-β-D-galactopyranoside (Compound S7) Compound S6 (49.4 g, 68.9 mmol) and Compound S4 (60 g, 1.5 equivalents) were dissolved in methylene chloride (500 mL), molecular sieves 4A (100 g) was added, and the mixture was stirred at room temperature for 30 minutes. 2,6-di-tert-butyl-4-methylpyridine (28 g, 2 equivalents) and dimethyl(methylthio)sulfonium triflate (50 wt% molecular sieves mixture, 71 g, 2 equivalents) were added, and the mixture was stirred at room temperature for 1 day. The reaction mixture was filtered through Celite, and the filtrate was washed with saturated aqueous sodium bicarbonate. Magnesium sulfate was added to the organic layer for drying. The desiccant was filtered off, and the solvent was evaporated under reduced pressure. The resulting residue was subjected to silica gel column chromatography (elution solvent: toluene:ethyl acetate=16:1) to obtain compound S7 (49.8 g, yield 65%).
¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 2.37, 2.42 (2 s, 6 H, 2 COPhMe), 3.49 (d, 2 H, J = 6.4 Hz, H-6a, H-6'a), 3.63-3.71 (m, 4 H, H-3a, H-5a, H-3b, H-5b), 3.68 (s, 3 H, PhOMe), 3.75 (dd, 1 H, J = 5.1 Hz, 10.1 Hz, H-6b), 3.85 (dd, 1 H, J = 5.5 Hz, 10.1 Hz, H-6'b), 3.91 (dd, 1 H, J = 5.5 Hz, 9.7 Hz, H-6'), 4.04, 4.16 (2 d, 2 H, J = 15.1 Hz, COCH ₂ Cl), 4.28 (d, 1 H, J = 2.7 Hz, H-4a), 4.37-4.67 (7 d, 8 H, 4 CH ₂ Ph), 4.83 (d, 1 H, J = 7.7 Hz, H-1a), 5.06 (d, 1 H, J = 8.2 Hz, H-1b), 5.33 (dd, 1 H, J = 8.2 Hz, 10.1 Hz, H-2b), 5.42 (dd, 1 H, J = 7.7 Hz, 10.1 Hz, H-2a), 5.66 (d, 1 H, J = 3.6 Hz, H-4), 6.63, 6.76 (2 m, 4 H, PhOMe), 7.01-7.39 (m, aromatic), 7.77, 7.95 (2 d, 4 H, 2 ortho- of COPhMe).

[2] Synthesis of 5-tert-butyl-2-methylphenyl(3,6-di-O-benzyl-4-O-chloroacetyl-2-O-p-toluoyl-β-D-galactopyranosyl)-(1-4)-3,6-di-O-benzyl-1-thio-2-O-p-toluoyl-β-D-galactopyranoside (Compound S8) Compound S7 (25 g, 22.3 mmol) was dissolved in methylene chloride (500 mL), and 5-tert-butyl-2-methylbenzenethiol (20 mL, 5 equivalents) and boron trifluoride diethyl ether complex (1.4 mL, 0.5 equivalents) were added. The mixture was stirred at room temperature for 1 hour, and the reaction solution was poured into saturated aqueous sodium bicarbonate (1 L). The organic layer was dried over magnesium sulfate, the desiccant was filtered off, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: toluene:ethyl acetate=20:1) to obtain compound S8 (13.6 g, yield 52%).
¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 1.17 (s, 9 H, t-Bu), 2.08 (s, 3 H, PhMe), 2.38, 2.43 (2 s, 6 H, 2 COPhMe), 3.46 (d, 2 H, J = 6.9 Hz, H-6a, H-6'a), 3.56-3.70 (m, 5 H, H-3a, H-5a, H-3b, H-5b, H-6b), 3.79 (dd, 1 H, J = 6.4 Hz, 9.6 Hz, H-6'b), 4.04, 4.17 (2 d, 2 H, J = 15.1 Hz, COCH ₂ Cl), 4.31 (d, 1 H, J = 2.3 Hz, H-4a), 4.35-4.67 (m, 8 H, 4 CH ₂ Ph), 4.58 (d, 1 H, J = 10.1 Hz, H-1a), 5.09 (d, 1 H, J = 7.8 Hz, H-1b), 5.32 (m, 2 H, H-2a, H-2b), 5.66 (d, 1 H, J = 3.2 Hz, H-4), 6.95-7.44 (m, aromatic), 7.77, 7.95 (2 d, 4 H, 2 ortho- of COPhMe).

[3] Synthesis of 4-methoxyphenyl (3,6-di-O-benzyl-2-O-p-toluoyl-β-D-galactopyranosyl)-(1-4)-3,6-di-O-benzyl-2-O-p-toluoyl-β-D-galactopyranoside (Compound S9) Compound S7 (20 g, 17.8 mmol) was dissolved in ethanol (800 mL), and 1,4-diazabicyclo[2.2.2]octane (20 g, 10 equivalents) was added, followed by stirring at 50°C for 2 hours. The reaction solution was cooled to room temperature, diluted with ethyl acetate, and washed with 2M hydrochloric acid and saturated aqueous sodium bicarbonate. Magnesium sulfate was added to the organic layer for drying, and the desiccant was filtered off. The solvent was then evaporated under reduced pressure to obtain Compound S9 (18.9 g, yield quant.).
¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 2.37, 2.42 (2 s, 6 H, 2 COPhMe), 2.63 (s, 1 H, OH), 3.56-3.88 (m, 8 H, H-3a, H-5a, H-6a, H-6'a, H-3b, H-5b, H-6b, H-6'b), 3.68 (s, 3 H, PhOMe), 4.06 (br. s, 1 H, H-4b), 4.38 (d, 1 H, J = 2.7 Hz, H-4a), 4.41-4.66 (m, 8 H, 4 CH ₂ Ph), 4.82 (d, 1 H, J = 7.8 Hz, H-1a), 5.13 (d, 1 H, J = 8.2 Hz, H-1b), 5.45 (m, 2 H, H-2a, H-2b), 6.62, 6.76 (2 m, 4 H, PhOMe), 7.03-7.39 (m, aromatic), 7.81, 8.00 (2 d, 4 H, 2 ortho- of COPhMe).

Example F3: Preparation of Gal(4mer) derivative

[1] Synthesis of 4-methoxyphenyl(3,6-di-O-benzyl-4-O-chloroacetyl-2-O-p-toluoyl-β-D-galactopyranosyl)-(1-4)-(3,6-di-O-benzyl-2-O-p-toluoyl-β-D-galactopyranosyl)-(1-4)-(3,6-di-O-benzyl-2-O-p-toluoyl-β-D-galactopyranosyl)-(1-4)-3,6-di-O-benzyl-2-O-p-toluoyl-β-D-galactopyranoside (Compound S10) Compound S8 (16.4 g, 12.5 mmol) and Compound S9 (18.9 g, 1.4 equivalents) were dissolved in methylene chloride (200 mL), molecular sieves 4A (40 g) was added, and the mixture was stirred at room temperature for 30 minutes. 2,6-Di-tert-butyl-4-methylpyridine (5.15 g, 2 equivalents) and dimethyl(methylthio)sulfonium triflate (50 wt% molecular sieves mixture, 12.9 g, 2 equivalents) were added and stirred at room temperature for 1 day. The reaction solution was filtered through Celite, and the filtrate was washed with saturated aqueous sodium bicarbonate. Magnesium sulfate was added to the organic layer for drying, and the desiccant was filtered off, after which the solvent was evaporated under reduced pressure. The resulting residue was subjected to silica gel column chromatography (elution solvent: toluene:ethyl acetate=15:1) to obtain compound S10 (20.0 g, yield 78%).
¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 2.19, 2.30, 2.37, 2.41 (4 s, 12 H, 4 COPhMe), 3.21-3.77 (m, 16 H), 3.69 (s, 3H, PhOMe), 3.94, 4.05 (2 d, J = 15.1 Hz, COCH ₂ Cl), 4.12 (d, 1 H, J = 2.8 Hz, H-4), 4.21-4.65 (m, 18 H), 4.71, 4.85, 5.10, 5.32 (4 d, 4 H, H-1), 5.20-5.35 (m, 4 H, H-2), 5.66 (d, 1 H, J = 3.2 Hz, H-4), 6.63, 6.75 (2 m, 4 H, PhOMe), 6.96-7.40 (m, aromatic), 7.74, 7.90, 7.97 (3 d, 8 H, 4 ortho- of COPhMe).

[2] Synthesis of benzyl (3,6-di-O-benzyl-4-O-chloroacetyl-2-O-p-toluoyl-β-D-galactopyranosyl)-(1-4)-(3,6-di-O-benzyl-2-O-p-toluoyl-β-D-galactopyranosyl)-(1-4)-(3,6-di-O-benzyl-2-O-p-toluoyl-β-D-galactopyranosyl)-(1-4)-3,6-di-O-benzyl-2-O-p-toluoyl-β-D-galactopyranoside (Compound S13) Compound S10 (11 g, 5.39 mmol) was dissolved in acetonitrile (110 mL) and toluene (82 mL) and cooled to 0°C. A solution of ammonium cerium(IV) nitrate (20 g, 7 equivalents) in water (55 mL) was added, and the mixture was stirred at 0°C for 3 hours. The reaction solution was extracted with ethyl acetate and washed with water, saturated aqueous sodium bicarbonate, and saturated brine, in that order. Magnesium sulfate was added to the organic layer to dry it. The desiccant was filtered off, and the solvent was evaporated under reduced pressure. The resulting residue was subjected to silica gel column chromatography (elution solvent: toluene:ethyl acetate=8:1) to obtain a crude product of compound S11 (10 g). This was dissolved in methylene chloride (100 mL) and cooled to 0°C. Trichloroacetonitrile (4.5 mL, 10 equivalents) and 1,8-diazabicyclo[5.4.0]-7-undecene (0.34 mL, 0.5 equivalents) were added, and the mixture was stirred at 0°C for 3 hours. The reaction solution was subjected to silica gel column chromatography (elution solvent: toluene:ethyl acetate=15:1) to obtain a crude product of compound S12 (9.5 g). This was dissolved in methylene chloride (60 mL), and benzyl alcohol (9.4 mL, 10 equivalents) and molecular sieves 4A (20 g) were added. After stirring at room temperature for 30 minutes, trimethylsilyl triflate (170 μL, 0.2 equivalents) was added and the mixture was stirred at room temperature for 4 hours. The reaction mixture was filtered through Celite, and the filtrate was washed with saturated aqueous sodium bicarbonate. The organic layer was dried over magnesium sulfate, and the desiccant was filtered off. The solvent was then evaporated under reduced pressure. The resulting residue was subjected to silica gel column chromatography (elution solvent: hexane:ethyl acetate = 2:1) to obtain compound S13 (8.2 g, yield 75%).
¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 2.32, 2.33, 2.43 (3 s, 12 H, 4 COPhMe), 3.25-3.77 (m, 16 H), 3.94, 4.05 (2 d, J = 15.1 Hz, COCH ₂ Cl), 4.16-4.65 (m, 22 H), 4.93, 5.07(2 d, 2 H, H-1), 5.17-5.35 (m, 5 H, H-1, H-2), 5.66 (d, 1 H, J = 3.2 Hz, H-4), 6.95-7.40 (m, aromatic), 7.75, 7.89, 7.91, 7.98 (4 d, 8 H, 4 ortho- of COPhMe).

Example F4: Sugar Chain Extension [1] Preparation of Gal Oligomer Acceptor

Gal oligomer derivatives (compounds S13, S15, S17, S19, S21, S23, S25, S27, and S29) were dissolved in ethyl acetate (7.5 mL/g of acceptor) and ethanol (7.5 mL/g of acceptor), and 1,4-diazabicyclo[2.2.2]octane (10 equivalents) was added and stirred at 50°C for 2 hours. The reaction solution was cooled to room temperature, diluted with ethyl acetate, and washed with 2M hydrochloric acid and saturated aqueous sodium bicarbonate. The organic layer was dried over magnesium sulfate, the desiccant was filtered off, and the solvent was evaporated under reduced pressure. The resulting residue was purified by silica gel column chromatography (elution solvent: toluene:ethyl acetate = 9:1) to obtain Gal oligomer acceptors (compounds S14, S16, S18, S20, S22, S24, S26, S28, and S30).
Compound S14:
Yield: 8.0g (yield: 89%). ¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 2.29-2.43 (4 s, 12 H, 4 COPhMe), 2.62 (br. s, 1 H, OH), 3.25-3.85 (m, 16 H), 4.06-4.65 (m, 23 H), 4.95, 5.15 (2 d, 2 H, H-1), 5.21-5.31 (m, 5 H, H-1, H-2), 5.44 (dd, 1 H, J = 8.2 Hz, 9.6 Hz, H-2), 6.94-7.38 (m, aromatic), 7.73-8.02 (4 d, 8 H, 4 ortho- of COPhMe).
Compound S16:
Yield: 7.7 g (59% yield from compound S14). ^1H -NMR (400 MHz, _CDCl3 ): δ (ppm) 2.23-2.43 (6 s, 18 H, 6 COPhMe), 2.63 (br. s, 1 H, OH), 3.27-3.85 (m, 24 H), 4.01-4.65 (m, 35 H), 4.96, 5.00 (2 d, 2 H, H-1), 5.08-5.30 (m, 8 H), 5.45 (dd, 1 H, J = 7.8 Hz, 9.6 Hz, H-2), 6.90-7.36 (m, aromatic), 7.73-8.02 (6 d, 12 H, 6 ortho- of COPhMe).
Compound S18:
Yield: 6.6 g (65% yield from compound S16). ¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 2.22-2.43 (7 s, 24 H, 8 COPhMe), 2.61 (br. s, 1 H, OH), 3.23-3.85 (m, 32 H), 4.05-4.64 (m, 45 H), 4.95, 4.96, 4.99, 5.04, 5.09 (5 d, 5 H, H-1), 5.07-5.30 (m, 9 H, H-1, H-2), 5.46 (dd, 1 H, J = 8.2 Hz, 9.7 Hz, H-2), 6.91-7.35 (m, aromatic), 7.73-8.02 (8 d, 16 H, 8 ortho- of COPhMe).
Compound S20:
Yield: 4.9 g (60% yield from compound S18). ¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 2.21-2.43 (9 s, 30 H, 10 COPhMe), 2.62 (br. s, 1 H, OH), 3.22-3.85 (m, 40 H), 4.02-4.64 (m, 55 H), 4.94-5.30 (m, 18 H, H-1, H-2), 5.45 (dd, 1 H, J = 8.2 Hz, 9.6 Hz, H-2), 6.91-7.35 (m, aromatic), 7.74-8.02 (m, 20 H, 10 ortho- of COPhMe). MALDI-TOF-MS: Calcd for C ₂₈₇ H ₂₈₈ O ₆₁ Na m/z [M+Na] ⁺ : 4732.9, Found: 4732.5.
Compound S22:
Yield: 3.1 g (52% yield from compound S20). ¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 2.21-2.43 (8 s, 36 H, 12 COPhMe), 2.63 (br. s, 1 H, OH), 3.22-3.85 (m, 48 H), 4.02-4.64 (m, 65 H), 4.93-5.30 (m, 22 H, H-1, H-2), 5.46 (dd, 1 H, J = 8.2 Hz, 10.1 Hz, H-2), 6.90-7.35 (m, 161 H, Ph), 7.74-8.02 (m, 24 H, 12 ortho- of COPhMe). MALDI-TOF-MS: Calcd for C ₃₄₃ H ₃₄₄ O ₇₃ Na m/z [M+Na] ⁺ : 5653.3, Found: 5651.4.
Compound S24:
Yield: 1.8 g (52% yield from compound S22). ¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 2.21-2.42 (9 s, 42 H, 14 COPhMe), 2.62 (br. s, 1 H, OH), 3.23-3.84 (m, 56 H), 4.02-4.64 (m, 75 H), 4.93-5.30 (m, 26 H, H-1, H-2), 5.46 (dd, 1 H, J = 8.2 Hz, 9.6 Hz, H-2), 6.90-7.36 (m, aromatic), 7.73-8.01 (m, 28 H, 14 ortho- of COPhMe). MALDI-TOF-MS: Calcd for C ₃₉₉ H ₄₀₀ O ₈₅ Na m/z [M+Na] ⁺ : 6572.7, Found: 6571.0.
Compound S26:
Yield: 800 mg (40% yield from compound S24). ¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 2.21-2.43 (7 s, 48 H, 16 COPhMe), 2.62 (br. s, 1 H, OH), 3.25-3.85 (m, 64 H), 4.02-4.65 (m, 85 H), 4.93-5.30 (m, 30 H, H-1, H-2), 5.46 (dd, 1 H, J = 8.3 Hz, 9.6 Hz, H-2), 6.90-7.34 (m, aromatic), 7.73-8.02 (m, 32 H, 16 ortho- of COPhMe). MALDI-TOF-MS: Calcd for C ₄₅₅ H ₄₅₆ O ₉₇ Na m/z [M+Na] ⁺ : 7499.5, Found: 7498.0.
Compound S28:
Yield: 360 mg (40% yield from compound S26). ¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 2.21-2.43 (8 s, 54 H, 18 COPhMe), 2.63 (br. s, 1 H, OH), 3.24-3.85 (m, 72 H), 4.02-4.65 (m, 95 H), 4.93-5.30 (m, 34 H, H-1, H-2), 5.46 (dd, 1 H, J = 7.8 Hz, 9.6 Hz, H-2), 6.90-7.34 (m, aromatic), 7.73-8.02 (m, 36 H, 18 ortho- of COPhMe). MALDI-TOF-MS: Calcd for C ₅₁₁ H ₅₁₂ O ₁₀₉ Na m/z [M+Na] ⁺ : 8420.6, Found: 8421.0.
Compound S30:
Yield: 210 mg (53% yield from compound S28). ¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 2.21-2.43 (6 s, 60 H, 20 COPhMe), 2.65 (br. s, 1 H, OH), 3.23-3.85 (m, 80 H), 4.02-4.65 (m, 105 H), 4.93-5.29 (m, 38 H, H-1, H-2), 5.46 (dd, 1 H, J = 7.8 Hz, 9.6 Hz, H-2), 6.90-7.34 (m, aromatic), 7.73-8.02 (m, 40 H, 20 ortho- of COPhMe). MALDI-TOF-MS: Calcd for C ₅₆₇ H ₅₆₈ O ₁₂₁ Na m/z [M+Na] ⁺ : 9341.7, Found: 9342.0.

[2] Glycosylation of Gal oligomer acceptor
Gal oligomer acceptors (compounds S14, S16, S18, S20, S22, S24, S26, and S28) and Gal (2mer) donors (compound 8, 3 equivalents) were dissolved in dichloromethane (10 mL/g of acceptor), molecular sieves 4A (4 g/g of acceptor) was added, and the mixture was stirred at room temperature for 4 hours. 2,6-di-tert-butyl-4-methylpyridine (6 equivalents) and dimethyl(methylthio)sulfonium triflate (50 wt% molecular sieves mixture, 6 equivalents) were added, and the mixture was stirred at room temperature for 1 day. The reaction solution was filtered through Celite, and the filtrate was washed with saturated aqueous sodium bicarbonate. The organic layer was dried over magnesium sulfate, and the desiccant was removed by filtration, after which the solvent was evaporated under reduced pressure. The resulting residue was subjected to silica gel column chromatography (eluent: toluene:ethyl acetate=11:1) to obtain crude purified products of Gal oligomer derivatives (compounds S15, S17, S19, S21, S23, S25, S27, and S29).
Compound S15:
¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 2.23-2.43 (5 s, 18 H, 6 COPhMe), 3.27-3.77 (m, 24 H), 3.93-4.65 (m, 34 H), 3.94, 4.05 (2 d, J = 14.7 Hz, COCH ₂ Cl), 4.95, 4.98, 5.04 (3 d, 3 H, H-1), 5.07-5.36 (m, 7 H, H-1, H-2), 5.34 (dd, 1 H, J = 8.2 Hz, 9.9 Hz, H-2), 5.66 (d, 1 H, J = 3.2 Hz, H-4), 6.90-7.40 (m, aromatic), 7.73-7.97 (6 d, 12H, 6 ortho- of COPhMe).
Compound S17:
¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 2.22-2.43 (7 s, 24 H, 8 COPhMe), 3.23-3.77 (m, 32 H), 3.94, 4.05 (2 d, J = 14.6 Hz, COCH ₂ Cl), 4.03-4.64 (m, 42 H), 4.95, 4.96, 4.99 (3 d, 3 H, H-1), 5.03-5.37 (m, 11 H, H-1, H-2), 5.35 (dd, 1 H, J = 7.8 Hz, 10.1 Hz, H-2), 5.67 (d, 1 H, J = 3.2 Hz, H-4), 6.91-7.40 (m, aromatic), 7.73-7.97 (8 d, 16 H, 8 ortho- of COPhMe).
Compound S19
¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 2.21-2.43 (9 s, 30 H, 10 COPhMe), 3.22-3.78 (m, 40 H), 3.95, 4.05 (2 d, J = 15.1 Hz, COCH ₂ Cl), 4.02-4.65 (m, 52 H), 4.93-5.30 (m, 18 H, H-1, H-2), 5.35 (dd, 1 H, J = 7.8 Hz, 9.7 Hz, H-2), 5.67 (d, 1 H, J = 3.7 Hz, H-4), 6.90-7.40 (m, aromatic), 7.74-7.97 (m, 20 H, 10 ortho- of MALDI-TOF-MS: Calcd for C ₂₈₉ H ₂₈₉ ClO ₆₂ Na m/z [M+Na] ⁺ : 4808.9, Found: 4808.8.
Compound S21:
¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 2.21-2.43 (8 s, 36 H, 12 COPhMe), 3.22-3.77 (m, 48 H), 3.94, 4.05 (2 d, J = 14.6 Hz, COCH ₂ Cl), 4.02-4.65 (m, 62 H), 4.93-5.30 (m, 22 H, H-1, H-2), 5.35 (dd, 1 H, J = 8.2 Hz, 10.1 Hz, H-2), 5.67 (d, 1 H, J = 3.2 Hz, H-4), 6.90-7.40 (m, aromatic), 7.74-7.97 (m, 24 H, 12 ortho- of MALDI-TOF-MS: Calcd for C ₃₄₅ H ₃₄₅ ClO ₇₄ Na m/z [M+Na] ⁺ : 5729.3, Found: 5728.9.
Compound S23:
¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 2.21-2.43 (8 s, 42 H, 14 COPhMe), 3.22-3.78 (m, 56 H), 3.94, 4.05 (2 d, J = 14.7 Hz, COCH ₂ Cl), 4.02-4.65 (m, 72 H), 4.93-5.37 (m, 26 H, H-1, H-2), 5.35 (dd, 1 H, J = 7.8 Hz, 9.6 Hz, H-2), 5.67 (d, 1 H, J = 2.7 Hz, H-4), 6.90-7.40 (m, aromatic), 7.74-7.97 (m, 28 H, 14 ortho- of MALDI-TOF-MS: Calcd for C ₄₀₁ H ₄₀₁ ClO ₈₆ Na m/z [M+Na] ⁺ : 6649.7, Found: 6648.8.
Compound S25:
¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 2.21-2.43 (8 s, 48 H, 16 COPhMe), 3.23-3.77 (m, 64 H), 3.94, 4.05 (2 d, J = 15.1 Hz, COCH ₂ Cl), 4.02-4.65 (m, 82 H), 4.93-5.30 (m, 30 H, H-1, H-2), 5.35 (dd, 1 H, J = 7.8 Hz, 9.7 Hz, H-2), 5.67 (d, 1 H, J = 2.8 Hz, H-4), 6.90-7.40 (m, aromatic), 7.73-7.97 (m, 32 H, 16 ortho- of MALDI-TOF-MS: Calcd for C ₄₅₇ H ₄₅₇ ClO ₉₈ Na m/z [M+Na] ⁺ : 7570.0, Found: 7570.5.
Compound S27:
¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 2.21-2.43 (8 s, 54 H, 18 COPhMe), 3.23-3.75 (m, 72 H), 3.97-4.66 (m, 94 H), 4.99-5.25 (m, 34 H, H-1, H-2), 5.35 (dd, 1 H, J = 8.3 Hz, 10.1 Hz, H-2), 5.65 (d, 1 H, J = 3.6 Hz, H-4), 6.92-7.39 (m, aromatic), 7.73-7.97 (m, 36 H, 18 ortho- of COPhMe). Calcd for C ₅₁₃ H ₅₁₃ ClO ₁₁₀ Na m/z [M+Na] ⁺ : 8497.1, Found: 8498.7.
Compound S29:
¹ H-NMR (400MHz, CDCl ₃ ): δ (ppm) 2.20-2.42 (7 s, 60 H, 20 COPhMe), 3.25-3.76 (m, 80 H), 3.97-4.65 (m, 104 H), 4.85-5.35 (m, 39 H, H-1, MALDI-TOF-MS: Calcd for C 569 H ₅₆₉ ClO _{122 Na} m/z [M+Na] ⁺ : 9418.1, Found: 9418.2.

Example F5: Deprotection
Compound S30 (200 mg, 21.5 μmol) was dissolved in tetrahydrofuran (4 mL) and methanol (8 mL), and lithium hydroxide monohydrate (90 mg, 2.15 mmol) was added. The mixture was stirred at 80°C for 3 days. The reaction mixture was diluted with ethyl acetate and washed with saturated brine. Magnesium sulfate was added to the organic layer, and the drying agent was removed by filtration. The solvent was evaporated under reduced pressure. The residue was subjected to silica gel column chromatography (eluent: toluene:ethyl acetate = 2:1) to obtain the deacylated product (110 mg). This product was dissolved in tetrahydrofuran (2 mL) and methanol (3 mL), and palladium/carbon (Pd 5%) (approximately 55% water-wet, 100 mg) was added. The mixture was stirred under a hydrogen atmosphere at room temperature for 3 days. Water (4 mL), methanol (2 mL), and tetrahydrofuran (2 mL) were added to the reaction mixture, and the mixture was stirred for an additional 4 days under a hydrogen atmosphere. The solid matter in the reaction mixture was filtered off, and the filtrate was concentrated under reduced pressure. The residue was subjected to gel filtration chromatography (Sephadex G-25, eluent: water) to obtain a crude purified product of Compound 31. The crude purified product was then subjected to reverse phase column chromatography (ISOLUTE (registered trademark) C18 (EC), eluent: water, 1% aqueous acetonitrile solution) to obtain Compound S31 (34 mg, yield 49%).
¹ H-NMR (400MHz, D ₂ O): δ (ppm) 3.39-3.79 (m, 100 H, 20 H, H-2, 3, 5, 6, 6'), 4.02 (m, 20 H, H-4), 4.63 (d, 19.6 H, J = 7.8 Hz, H-1β), 5.25 (d, 0.4 H, J = 3.4 Hz, H-1α). ¹³ C-NMR(100MHz, D ₂ O): δ (ppm) 104.94 (C-1), 78.22 (C-4), 75.08 (C-5), 73.87 (C-3), 72.38 (C-2), 61.31 (C-6). MALDI-TOF-MS: Calcd for C ₁₂₀ H ₂₀₂ O ₁₀₁ Na m/z [M+Na] ⁺ : 3282.1, Found: 3281.9.

The NMR analysis data was similar to the values derived from "→4)-β-D-Galp-(1→)" reported in the literature (Food Chemistry, 242, 211-216(2018)), supporting the idea that compound S31 has 20 galactose residues linked together in a β1,4 bond. Furthermore, MALDI-TOF MS analysis revealed peaks at [M+Na]+ 3281.91 and [M+K]+ 3297.87, which correspond to the molecular weight of 20 galactose sugars.

Example F6: Catalytic activity of polysaccharides prepared by organic synthesis 2 mg of compound S31 synthesized in Example F-5 was weighed into a 2 mL glass bottle, and 200 μL of a 25 mM aqueous sodium chloride solution was added, followed by lyophilization. The catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method III." The results are shown in Table 54. When compound S31 was compared with a BG3 fraction containing polysaccharides prepared from water-soluble soybean polysaccharides in the same manner as in Example A7(6) and having an average molecular weight similar to that of compound S31, the catalytic activity and stereoselectivity were found to be comparable or higher.

Claims (9)

A polysaccharide having a degree of polymerization of 16 to 40 and consisting solely of galactose,
A polysaccharide in which sugar units are linked in a β1,4 bond in an unbranched chain. A polysaccharide having a degree of polymerization of 16 to 40, consisting only of galactose, and having β1,4 bonds in an unbranched chain ,
A polysaccharide in which the terminal galactose of the polysaccharide is a reductively aminated amino sugar. A composition comprising the polysaccharide of claim 1 or 2 . 4. The composition of claim 3 for use as a catalyst. A method for producing the polysaccharide according to claim 1 or 2, comprising:
a step of mixing the processed plant product with an organic solvent and filtering out the solid matter to obtain a defatted sample;
mixing the defatted sample with water to remove soluble polysaccharides;
and heating the sample from which the soluble polysaccharides have been removed under acidic conditions, or in the presence of EDTA or ammonium oxalate to obtain crude pectic polysaccharides . A compound represented by formula (1) in the presence of the polysaccharide according to claim 1 or 2 ,

wherein X is —O—, —NR—, or —S—;
R is a hydrogen atom, an optionally substituted C _1-6 alkyl group, an optionally substituted C _3-6 cycloalkyl group, an optionally substituted C 2-6 alkenyl group, an optionally substituted C _3-6 cycloalkenyl group, an optionally substituted C _2-6 alkynyl group, an optionally substituted C _6-10 aryl group, an optionally substituted C _1-6 alkylcarbonyl group, an optionally substituted C _6-10 arylcarbonyl group, an optionally substituted C _1-6 alkylsulfonyl group or _{a C 6-10 arylsulfonyl} group;
R ¹ , R ² , R ³ and R ⁴ are each independently a hydrogen atom, an optionally substituted C _1-6 alkyl group, an optionally substituted C _3-6 cycloalkyl group, an optionally substituted C _2-6 alkenyl group, an optionally substituted C _3-6 cycloalkenyl group, an optionally substituted C _2-6 alkynyl group or an optionally substituted C _6-10 aryl group;
^R2 and ^R3 may be bonded to each other to form a cyclic structure represented by (1a).

wherein X is —O—, —NR—, or —S—;
R ¹ and R ⁴ are each independently a hydrogen atom, an optionally substituted C _1-6 alkyl group, an optionally substituted C _3-6 cycloalkyl group, an optionally substituted C _2-6 alkenyl group, an optionally substituted C _3-6 cycloalkenyl group, an optionally substituted C _2-6 alkynyl group, or an optionally substituted C _6-10 aryl group;
R ^2a and R ^3a are each independently a C _1-6 alkylene group, a C _1-6 oxyalkylene group, a C _3-6 cycloalkylene group, a C _2-6 alkenylene group, a C _3-6 cycloalkenylene group, a C _2-6 alkynylene group, or a C _6-10 arylene group.]
reacting a compound represented by formula (2) with

wherein Y is —O—, —NR ⁶ — or —S—;
R ⁵ and R ⁶ are each independently a hydrogen atom, an optionally substituted C _1-6 alkyl group, an optionally substituted C _3-6 cycloalkyl group, an optionally substituted C _2-6 alkenyl group, an optionally substituted C _3-6 cycloalkenyl group, an optionally substituted C _2-6 alkynyl group, or an optionally substituted C _6-10 aryl group;
^R5 and ^R6 may be bonded to each other to form a cyclic structure represented by (2a),
However, the compound represented by formula (2) is not water or hydrogen sulfide.]

[In the formula, R ^5a and R ^6a each represent an optionally substituted C _1-6 alkylene group, an optionally substituted C _1-6 oxyalkylene group, an optionally substituted C _3-6 cycloalkylene group, an optionally substituted C _2-6 alkenylene group, an optionally substituted C _3-6 cycloalkenylene group, an optionally substituted C _2-6 alkynylene group, or a C _6-10 arylene group.]
A method for producing a compound represented by formula (3), comprising the step of obtaining a compound represented by formula (3).

[In the formula, X, Y, R ¹ , R ² , R ³ , R ⁴ and R ⁵ are as defined in formula (1) or (2)] (1) reacting 7-oxabicyclo[4.1.0]heptane and cyclopropylamine in the presence of a polysaccharide according to claim 1 or 2 to obtain (1R,2R)-2-cyclopropylamino-1-cyclohexanol;
(2a) reacting (1R,2R)-2-cyclopropylamino-1-cyclohexanol with a carbonylation reagent, followed by further reaction with protected or unprotected 6-(3-aminopropoxy)-2(1H)-quinolinone, or (2b) reacting protected or unprotected 6-(3-aminopropoxy)-2(1H)-quinolinone with a carbonylation reagent, followed by further reaction with (1R,2R)-2-cyclopropylamino-1-cyclohexanol, in which, when protected 6-(3-aminopropoxy)-2(1H)-quinolinone is used, further deprotection is carried out to obtain (-)-6-[3-[3-cyclopropyl-3-[(1R,2R)-2-hydroxycyclohexyl]ureido]-propoxy]-2(1H)-quinolinone;
A method for producing (-)-6-[3-[3-cyclopropyl-3-[(1R,2R)-2-hydroxycyclohexyl]ureido]propoxy]-2(1H)-quinolinone, comprising: The method of claim 5, further comprising purifying the crude pectic polysaccharides by treatment with an enzyme, alkali, or acid, fractionation by adding an organic solvent, fractionation utilizing temperature-dependent solubility differences, treatment with an anion exchange resin, gel filtration chromatography, HPLC , or any combination thereof. A method for producing a galactan in which 16 to 24 galactoses are β1,4-linked, comprising:
A method comprising the step of reacting a compound represented by formula (14) with a compound represented by formula (16) to obtain a compound represented by formula (17).

[In the formula, R ^A is an optionally substituted alkylcarbonyl group, R ^B is an optionally substituted aralkyl group, R ^C is an optionally substituted arylcarbonyl group, R ^D is an optionally substituted aryl group, R ^E is an optionally substituted aralkyl group, and n is an integer from 13 to 21. ]