Plant-derived organic molecular catalysts

Abstract

The present invention provides a polysaccharide which has a degree of polymerisation of 16-40 and is composed only of galactose or composed only of galactose and 1-3 molecules of arabinose, the saccharide units of said polysaccharide being bonded in an unbranched chain.

Description

[Technical Field]

[0001] The present invention relates to a plant-derived organocatalyst and a method for producing the same. [Background technology]

[0002] 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 there are at least two asymmetric carbon atoms in one molecule, multiple optical isomers exist. 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 transition metal catalysts used in asymmetric reactions. [ka] In the formula, X is —O— or —NR f -, and Y represents -O-, -NR g - or -S-, R a , R b , R c , R d , R e , R f and R g each independently represents a hydrogen atom or an organic group, and an asterisk indicates that the carbon atom is an asymmetric carbon.

[0003] 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-).

[0004] 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 compound having an epoxide structure or an aziridine structure, which are readily available, with a nucleophile and stereoselectively opening the ring is highly atom-efficient and useful.

[0005] For example, methods for obtaining optically active compound (Z) by reacting a compound having an epoxide structure with a nucleophile include (A) a method of optically resolving a racemate (e.g., Patent Documents 1 to 3, Non-Patent Documents 1 and 2), (B) a method of introducing an asymmetric carbon atom into another position and separating the resulting diastereomers (e.g., Patent Document 4, Non-Patent Document 3), and (C) a method of 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 to 6). In particular, known methods for 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 an enzyme derived from an animal or a microorganism.

[0006] 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 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).

[0007] However, in the methods (A), (B) and (D), the theoretical yield does not exceed 50%, and the use of a resolving agent in an amount equal to the product or purification using a large-capacity column is required, which poses a problem as an industrial production method.

[0008] On the other hand, methods (C) and (E) 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 recovery step for the expensive metal catalyst.

Prior Technical Literature

Charter Documents

[0009] [Patent Document 1] Patent No. 4406483 [Patent Document 2] Patent No. 4406482 [Patent Document 3] U.S. Patent No. 5981267 [Patent Document 4] Special Publication No. 9-157258 [Patent Document 5] Japanese Patent Publication No. 2003-206266 [Patent Document 6] Japanese Patent Publication No. 2011-83934 [Patent Document 7] Patent Gazette No. 6630667 [Patent Document 8] Patent Gazette No. 6678442

Non-licensed literature

[0010]

Non-patent document 1

Non-patent document 2

Non-patent document 3

Non-patent document 4

Non-patented document 5

Non-patent document 6

Non-patent document 7

[0011] The present inventors have previously found that the asymmetric ring-opening reaction of epoxides or aziridines with nucleophiles proceeds in the presence of processed plant products (Patent Document 7, Non-Patent Documents 10 and 11). They also found that the catalytic activity of processed plant products is improved by treating them with an enzyme (Patent Document 8).

[0012] However, because the chemical structure of the catalyst's active form is unknown, it is difficult to obtain a more highly active catalyst, and it has been noticed 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 the water-soluble soybean polysaccharide SOYAFIBE S-DN is 100,000 to 850,000 (Non-Patent Document 9)), which has been found to pose handling problems, 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 gelation or adhesion of the catalyst to the container reduces the amount of product recovered. 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. [Means for solving the problem]

[0013] The present inventors have discovered the active substance of the catalyst by removing lipids and soluble polysaccharides from processed plant products to obtain crude pectic polysaccharides, and then repeatedly treating and purifying the crude pectic polysaccharides with enzymes (e.g., lipase, galactanase), acids, alkalis, or ion exchange resins, thereby completing the present invention.

[0014] That is, the present invention provides the following [1] to

[30] . [1] A polysaccharide with 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 of 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, bound in an unbranched chain, which is composed solely of galactose, or a polysaccharide composed solely of galactose and 1 to 3 molecules of arabinose, 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 the asymmetric ring-opening reaction of a compound having an oxirane, aziridine or thiirane ring with a nucleophile.

[10] Mixing the processed plant product with an organic solvent, filtering the solid matter, 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 crude pectic polysaccharides obtained by the method described in

[10] as a catalyst or raw material for a catalyst.

[12] The use according to

[11] , wherein the catalyst promotes the asymmetric ring-opening reaction of a compound having an oxirane, aziridine or thiirane ring with a nucleophile.

[13] 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 galactose units bonded in a β1,4 bond, a compound represented by formula (1); [ka] wherein X is —O—, —NR—, or —S—; R is a hydrogen atom, an optionally substituted C 1-6alkyl group, optionally substituted C 3-6 Cycloalkyl groups, optionally substituted C 2-6 Alkenyl group, optionally substituted C 3-6 Cycloalkenyl group, optionally substituted C 2-6 Alkynyl group, optionally substituted C 6-10 Aryl group, optionally substituted C 1-6 Alkylcarbonyl group, optionally substituted C 6-10 Arylcarbonyl group, optionally substituted C 1-6 Alkylsulfonyl group or C 6-10 is an 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, optionally substituted C 3-6 Cycloalkyl groups, optionally substituted C 2-6 Alkenyl group, optionally substituted C 3-6 Cycloalkenyl group, optionally substituted C 2-6 an alkynyl group or an optionally substituted C 6-10 is an aryl group, R 2 and R 3 may be bonded to each other to form a cyclic structure.] reacting a compound represented by formula (2) with [ka] 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, optionally substituted C 3-6 Cycloalkyl groups, optionally substituted C 2-6 Alkenyl group, optionally substituted C 3-6 Cycloalkenyl group, optionally substituted C 2-6an alkynyl group or an optionally substituted C 6-10 is an aryl group, R 5 and R 6 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). [ka] [where X, Y, R 1 , R 2 , R 3 , R 4 and R 5 are the same as defined above.]

[14] The method according to

[13] , wherein the compound represented by formula (1) is a compound represented by formula (1a). [ka] 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, optionally substituted C 3-6 Cycloalkyl groups, optionally substituted C 2-6 Alkenyl group, optionally substituted C 3-6 Cycloalkenyl group, optionally substituted C 2-6 an alkynyl group or an optionally substituted C 6-10 is an aryl group, R 2a and R 3a are each independently C 1-6 Alkylene group, C 1-6 Oxyalkylene group, C 3-6 Cycloalkylene group, C 2-6 Alkenylene group, C 3-6 Cycloalkenylene group, C 2-6 Alkynylene group or C 6-10 is an arylene group.]

[15] The method according to

[13] or

[14] , wherein the compound represented by formula (2) is a compound represented by formula (2a): [ka] [In the formula, R 5a and R 6a are optionally substituted C 1-6 Alkylene group, optionally substituted C 1-6 Oxyalkylene group, optionally substituted C 3-6 Cycloalkylene group, optionally substituted C 2-6 Alkenylene group, optionally substituted C 3-6 Cycloalkenylene group, optionally substituted C 2-6 Alkynylene group or C 6-10 is an 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 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 galactose units linked in a β1,4 bond, at least one of the compounds represented by formula (a) to (b) is reacted with a galactan having 16 to 24 galactose units linked in a β1,4 bond. [ka] [In the formula, R 1 , R 2 , R 3 and R 4 are each independently a hydrogen atom, an optionally substituted C 1-6 alkyl group, optionally substituted C 3-6 Cycloalkyl groups, optionally substituted C 2-6 Alkenyl group, optionally substituted C 3-6 Cycloalkenyl group, optionally substituted C 2-6 an alkynyl group or an optionally substituted C 6-10 is an aryl group, R 2 and R 3may be bonded to each other to form a cyclic structure.] reacting a compound represented by formula (2) with [ka] 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, optionally substituted C 3-6 Cycloalkyl groups, optionally substituted C 2-6 Alkenyl group, optionally substituted C 3-6 Cycloalkenyl group, optionally substituted C 2-6 an alkynyl group or an optionally substituted C 6-10 is an aryl group, R 5 and R 6 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). [ka] [In the formula, Y, R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are the same as defined above.]

[18] (1) a step of reacting 7-oxabicyclo[4.1.0]heptane (also called 1,2-epoxycyclohexane) and cyclopropylamine 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 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 (1R,2R)-2-cyclopropylamino-1-cyclohexanol with a carbonylation reagent, followed by further reaction with protected or unprotected 6-(3-aminopropoxy)-2(1H)-quinolinone. 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 described in [3], comprising purifying a processed plant product or a crude pectic polysaccharide obtained by the method described in

[10] by treatment with an enzyme or alkali, fractionation by adding an organic solvent, or a combination of fractionation by adding 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 described in [2], comprising purifying a processed plant product or a crude pectic polysaccharide obtained by the method described in

[10] by enzymatic treatment, fractionation by adding an organic solvent, treatment with an anion exchange resin, gel filtration chromatography, or any combination thereof.

[22] A method for producing the polysaccharide described in [1], comprising purifying the polysaccharide described in [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] The use of galactans, which are composed of 16 to 24 β1,4-linked galactoses, as catalysts.

[25] The use according to

[24] , wherein the catalyst promotes the 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). [ka] [In the formula, R A is an optionally substituted alkylcarbonyl group, and R B is an optionally substituted aralkyl group, and R C is an optionally substituted arylcarbonyl group, and R D is an optionally substituted aryl group, and R E represents an optionally substituted aralkyl group, and n is an integer of 13 to 21.]

[27] R C

[26] The method according to

[26] , wherein

[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). [ka] [In the formula, R A is an optionally substituted alkylcarbonyl group, and R B is an optionally substituted aralkyl group, and R C is an optionally substituted arylcarbonyl group, and RF is an optionally substituted aryl group.

[29] In the presence of a Lewis acid, a compound represented by formula (13) and R D The method according to any one of

[26] to

[28] , further comprising a step of reacting SH to obtain a compound represented by formula (14). [ka] [In the formula, R A is an optionally substituted alkylcarbonyl group, and R B is an optionally substituted aralkyl group, and R C is an optionally substituted arylcarbonyl group, and 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). [ka] [In the formula, R A is an optionally substituted alkylcarbonyl group, and R B is an optionally substituted aralkyl group, and R C is an optionally substituted arylcarbonyl group, and R D is an optionally substituted aryl group, and R F is an optionally substituted aryl group. [Effects of the Invention]

[0015] According to the present invention, a new catalyst can be provided that can promote the asymmetric ring-opening reaction of epoxides or aziridines with nucleophiles. Furthermore, according to the present invention, by further purifying inexpensively available plant processed products, a catalyst with a higher specific activity than the catalysts disclosed in Patent Documents 7-8 and Non-Patent Documents 10-11 can be provided, which can also be reused. Furthermore, according to the present invention, the product and catalyst can be more easily recovered than in reactions using plant processed products. Furthermore, a catalyst can be provided that contains fewer impurities (e.g., allergens) that are problematic in pharmaceutical production. [Brief explanation of the drawings]

[0016] [Figure 1] 1 shows photographs of SDS-PAGE gels of fractions in each purification step of Example A3. [Figure 2] 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. [Figure 3] 1 is a chromatogram showing the time course of digestion of LP-LMW with Enzyme A in Example A6. [Figure 4] 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). [Figure 5] Graph (a) shows the catalytic activity of each NG fraction in Example A9, and graph (b) shows the stereoselectivity of each NG fraction in Example A9. [Figure 6] 1 is a graph showing the arabinose content of each NG fraction in Example A9. [Figure 7] 1(a) is a chromatogram of "fraction NG 3-4 kDa" obtained by HILIC-HPLC in Example A10, and FIG. 1(b) is a graph showing the chromatograms of each fraction after separation. [Figure 8] (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 the chromatograms of HILIC-HPLC analysis of each fraction after separation, superimposed on each other. [Figure 9] 1 is a graph showing the catalytic activity and stereoselectivity of fractions corresponding to the major peak and minor peak in Example A10(5). [Figure 10] 1 is a chromatogram of ODS-HPLC fractionation in Example A10(5). The dashed squares indicate fractionated peaks. [Figure 11] 1 is a graph showing superimposed chromatograms of ODS-HPLC analysis of each sample after ODS-HPLC fractionation in Example A10(6). [Figure 12] 1 is a chromatogram of ODS-HPLC preparative in Example A11. [Figure 13] 1 is a graph showing a chromatogram of ODS-HPLC analysis performed before ODS-HPLC fractionation in Example A11 superimposed on a chromatogram of ODS-HPLC analysis of each sample obtained by fractionating each of peaks C, D, E, F, G, H, and I. [Figure 14] 1(a) is a graph showing the catalytic activity of DP16 to DP25 in Example A11, and FIG. 1(b) is a graph showing the stereoselectivity of DP16 to DP25 in Example A11. [Figure 15] (a) is a chromatogram showing the results of HPLC analysis (RI detection) of NG-ABEE and the sample (NG) before modification in Example A12. (b) is a chromatogram showing the results of HPLC analysis (UV detection) of NG-ABEE and the sample (NG) before modification in Example A12. [Figure 16] 1H-NMR spectrum of AL-LMW in Example B1. [Figure 17]1 is a 13C-NMR spectrum of AL-LMW in Example B1. [Figure 18] 1 shows GC spectra obtained by detecting m / z 118 after derivatization of 19.5mer and 20mer in the analysis of sugar chain binding positions in Example B2. [Figure 19] 1 is a photograph showing the state of each reaction vessel before the toluene layer is collected in Example C4. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention will be described in detail below.

[0018] In this specification, galactose is also referred to as "Gal" and arabinose as "Ara."

[0019] As used herein, "unbranched chain" refers to a state in which each sugar unit (galactose, arabinose) except for the terminal sugar unit is bonded to two adjacent sugar molecules.

[0020] 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.

[0021] The polysaccharide according to this embodiment may be a galactan consisting of 16 to 40 galactose molecules, or may be an arabinogalactan in which 1 to 3 of the 16 to 40 galactose molecules are replaced with arabinose.

[0022] 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, fractionating by adding organic solvents, fractionating by cooling to exploit differences in solubility, or purifying by column chromatography (e.g., anion exchange, gel filtration, HPLC). The polysaccharides of this embodiment may also be artificially synthesized using organic chemistry techniques.

[0023] Second Embodiment The second embodiment of the present invention is a polysaccharide comprising the polysaccharide of 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.

[0024] The polysaccharide according to this embodiment may have a sugar composition containing 90 to 99.5 mol % of galactose and 0.5 to 10 mol % of arabinose, and may be composed of only galactose and arabinose, or may further contain other monosaccharides such as rhamnose, fructose, xylose, glucose, galacturonic acid, etc. Galactose, arabinose, and other monosaccharides may each independently be either a furanose type or a pyranose type.

[0025] The polysaccharide according to this embodiment has an average molecular weight of 2,500 to 9,000, preferably 2,700 to 8,000, and more preferably 2,900 to 7,000.

[0026] 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.

[0027] The polysaccharide according to this embodiment may have a sugar composition containing 80 to 98 mol % of galactose and 2 to 20 mol % of arabinose, and may be composed of only galactose and arabinose, or may further contain other monosaccharides such as rhamnose, fructose, xylose, glucose, galacturonic acid, etc. Galactose, arabinose, and other monosaccharides may each independently be either a furanose type or a pyranose type.

[0028] The polysaccharide according to this embodiment has an average molecular weight of 10,000 to 60,000, and preferably 11,000 to 55,000.

[0029] The polysaccharides according to the first to third embodiments can be produced as follows. For more specific details, the examples of the present application may be referred to. In the procedures described below, the method of removing insoluble matter or obtaining a precipitate by centrifugation may be replaced by decantation or filtration (natural filtration, centrifugal filtration, pressure filtration, or reduced pressure filtration). Furthermore, the method of purifying using an anion exchange resin column in the method described below may be performed by batchwise adding anion exchange resin to a sample before purification, followed by filtration.

[0030] (1) Degreasing process 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, thereby obtaining a defatted sample. To more efficiently remove fat-soluble components, the processed plant product is preferably pulverized into powder. Examples of organic solvents include halogenated hydrocarbons such as dichloromethane and chloroform, alcohols such as methanol and ethanol, and mixtures of these solvents. The temperature in the degreasing step may be room temperature (e.g., 0°C to 30°C).

[0031] <Plant processed products> In this specification, the processed plant product refers to a powder or extract obtained by processing a part of a plant.

[0032] The term "plant" as used above, in the case of edible plants, 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, beans, vegetables, fruits, or potatoes, and the parts of edible plants can be appropriately selected from the whole fruit, pulp, pericarp, stems (stalk-like parts in the case of algae), seeds, germs, roots (appressorium in the case of algae), bulbs, and leaves. In addition, the remainder (inedible parts) after harvesting the edible parts can also be considered as 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), 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 those from the following families: Onion, garlic, and leek, Zingiberaceae (e.g., spring turmeric), Palmaceae (e.g., coconut), Cupressaceae (e.g., juniper berry), Equisetaceae (e.g., horsetail), Rutaceae (e.g., summer mandarins, yuzu, flowered yuzu, and pomelo), Nelumbaceae (e.g., lotus root), Actinidiaceae (e.g., kiwi), Sargassumaceae (e.g., hijiki), Bocconiaceae (e.g., nori), and Funoriaceae (e.g., funori).The edible plants are preferably selected from the group consisting of: Magnoliaceae, 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. Furthermore, the "plant" may also be a non-edible plant, such as tobacco.

[0033] The term "processing" as used herein means, as needed, drying, heating, roasting, frying, fermenting, removing unnecessary parts, or the like, followed by grinding into powder or extracting components. The processed plant product also includes powder obtained by extracting an extract from an edible plant and then grinding the dried product. Therefore, the tea may be green tea or black tea. The soybean may be soybean flour or natto.

[0034] The processed plant products may be commercially available in a powder or liquid form, or may be commercially available processed products that are appropriately pulverized into powder and used. 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 soybean pulp are preferably used, and soybean flour, Soya Flour FT-N, Soya Five S-DN, or soybean pulp are more preferably used.

[0035] (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. The water can be tap water, purified water, or RO water (water filtered through a reverse osmosis membrane). The temperature in this step can be room temperature or heated (e.g., 0°C to 50°C). The centrifugal force used in centrifugation can be, for example, 3,000 to 20,000 × g.

[0036] (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 mixture 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, which is then stirred and centrifuged 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.

[0037] 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 processed product options available for use as catalysts. Furthermore, even 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.

[0038] (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 of the third embodiment.

[0039] (5) Alkali treatment of processed plant products A processed plant product or a crude pectic polysaccharide produced from a processed plant product is heated to 50 to 100°C in the presence of an alkali (e.g., sodium hydroxide, potassium hydroxide) and stirred, 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.

[0040] 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×g.

[0041] (6) Low molecular weight LP-LMW or AL-LMW (6-1) Depolymerization of polysaccharides by galactanase The LP-LMW or AL-LMW obtained in steps (4) and (5) above is purified using an anion exchange resin column (OH type), and the fraction eluted earlier is collected. The resulting fraction (LMW-D1) may correspond to the polysaccharide of the third embodiment.

[0042] 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 resulting polysaccharide is purified using an anion exchange resin column (OH type), and the earlier eluted fraction is collected. The resulting polysaccharide (LMW-D1degD1) may correspond to the polysaccharide of the second embodiment.

[0043] (6-2) Depolymerization of Polysaccharides by Acid The polysaccharide (LP-LMW or AL-LMW) obtained in steps (4) and (5) above is 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) or 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, or 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.

[0044] (6-3) Depolymerization of Polysaccharides by Cation Exchange Resin The polysaccharide (LP-LMW or AL-LMW) obtained in steps (4) and (5) above is dissolved in water, and a cation exchange resin is added (pH is approximately 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.

[0045] (6-4) Direct synthesis of low molecular weight compounds from processed plant products A processed plant product or crude pectic polysaccharide produced from the processed plant product is dissolved in water, heated in an autoclave, and then centrifuged to remove insoluble matter. The resulting supernatant is purified by anion exchange 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 reduced molecular weight polysaccharide (SFDdegE2 fraction). The resulting polysaccharide is purified by anion exchange column chromatography to obtain a reduced molecular weight polysaccharide (SFDdegE2D1 fraction). This is further purified by dialysis and gel filtration column chromatography to obtain a reduced molecular weight polysaccharide (NG fraction). The resulting polysaccharides (SFDdegE2, SFDdegE2D1, and NG fractions) may correspond to the polysaccharides of the second embodiment.

[0046] Fourth Embodiment The catalyst according to the present invention may be a galactan in which 16 to 24 galactose units are linked via 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. [ka]

[0047] The galactan according to this embodiment can be produced by a method known in the field of organic chemical synthesis. For example, compound (17) produced by the following method is deprotected (i.e., R A , R B , R C and R E Compound (17) can be prepared by subjecting compound (14) and compound (16) to a glycosylation reaction. [ka]

[0048] R Ais an optionally substituted alkylcarbonyl group, for example, C 1-6 It is an alkylcarbonyl group. A is preferably an acetyl group, a chloroacetyl group, a dichloroacetyl group, a trichloroacetyl group, or a bromoacetyl group, and more preferably a trichloroacetyl group. A is also a protecting group for the hydroxyl group, and can be removed by hydrolysis under acidic or alkaline conditions.

[0049] R B and R E is an optionally substituted aralkyl group, for example, C 6-10 C substituted with aryl groups 1-6 R is an alkyl group. B is preferably C 1-6 Alkyl group, C 1-6 C is a benzyl group, a phenylethyl group, a 1-naphthylmethyl group, or a 2-naphthylmethyl group, each of which may be substituted with at least one selected from an alkoxy group and a halogen atom. 1-6 Examples of the 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. 1-6 Examples of the alkoxy group include a methoxy group, an ethoxy group, a 1-propyloxy group, a 2-propyloxy group, a 1-butyloxy group, a 2-butyloxy group, a tert-butyloxy group, a 1-pentyloxy group, a 2-pentyloxy group, a 3-pentyloxy group, a neopentyloxy group, a 1-hexyloxy group, a 2-hexyloxy group, and a 3-hexyloxy group. Examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom. R B is also a protecting group for a hydroxyl group and can be removed by subjecting to hydrogenolysis conditions. Hydrogenolysis conditions include, for example, reacting with hydrogen in the presence of palladium / carbon. The catalyst for reacting with hydrogen may be any catalyst that can be used in hydrogenolysis.

[0050] R C is an optionally substituted arylcarbonyl group, for example, optionally substituted C 6-10 An example of such a group is an arylcarbonyl group. C is preferably C 1-6 Alkyl group, C 1-6 R is a phenylcarbonyl group which may be substituted with at least one selected from an alkoxy group and a halogen atom, and more preferably an ortho-, meta-, or para-toluenecarbonyl group (also called a toluoyl group). C When R 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 allows for selective formation of a β-bond. Furthermore, when synthesizing polysaccharides with eight or more monosaccharides, the physical properties of the raw materials and the product are similar, making it difficult to track the progress of the reaction at each step. C When R is an optionally substituted arylcarbonyl group, in addition to the selective glycosylation due to neighboring group participation described above, it becomes easier to follow the progress of the reaction. 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.

[0051] R D is an optionally substituted aryl group, for example, an optionally substituted C 6-10 Aryl is an example of R D is preferably C 1-6 Alkyl group, C 1-6 It is a phenyl group which may be substituted with at least one selected from an alkoxy group and a halogen atom, and more preferably a 5-tert-butyl-2-methylphenyl group.

[0052] n is an integer of 13 to 21, and preferably 15 to 19, or 17.

[0053] Compound (15) can be obtained by hydrolyzing compound (13). A The ester bond represented by O is broken. A Any conditions may be used as long as they allow hydrolysis of the ester bond represented by O. For example, acidic or alkaline conditions can be applied, with alkaline conditions being preferred. An example of alkaline conditions is adding 1,4-diazabicyclo[2.2.2]octane (DABCO) to a mixed solvent of ethyl acetate and ethanol. [ka] In the formula, R A is an optionally substituted alkylcarbonyl group, and R B is an optionally substituted aralkyl group, and R C is an optionally substituted arylcarbonyl group, and R F is an optionally substituted aryl group.

[0054] R F is an optionally substituted aryl group, for example, an optionally substituted C 6-10 Aryl is an example of R F is preferably C 1-6 Alkyl group, C 1-6 R is a phenyl group which may be substituted with at least one selected from an alkoxy group and a halogen atom, and more preferably a 4-methoxyphenyl group. F is also a protecting group for the hydroxyl group and can be removed by hydrolysis under acidic or alkaline conditions. F When R is a 4-methoxyphenyl group, R E The oxidative conditions include, for example, the addition of ammonium cerium(IV) nitrate (CAN) in a mixed solvent of acetonitrile, toluene, and water.

[0055] Compound (16a) was prepared by glycosylation of compound (16) with compound (14) to give compound (17), followed by ORA is obtained by converting the protecting group (R) to OH. By replacing compound (16) with compound (16a) and repeating these steps, two galactoses can be added to produce a longer galactan structure. That is, compound (18) can be obtained by removing the protecting group (R) from compound (17), which is produced by repeating these steps. A , R B , R C , R E ) can be produced by removing [ka]

[0056] In the glycosylation reaction using compound (14), an activating agent is added to activate compound (14). The activating agent can 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.

[0057] Compound (14) can be prepared by reacting compound (13) with R D It is obtained by glucosylating SH. [ka] In the formula, R A is an optionally substituted alkylcarbonyl group, and R B is an optionally substituted aralkyl group, and R C is an optionally substituted arylcarbonyl group, and R D is an optionally substituted aryl group, and R F is an optionally substituted aryl group.

[0058] The Lewis acid may be any acid capable of activating the anomeric position, such as boron trifluoride diethyl ether complex.

[0059] Compound (13) can be obtained by glycosylation of compound (11) and compound (12) in the presence of a Lewis acid. [ka] In the formula, R A is an optionally substituted alkylcarbonyl group, and R B is an optionally substituted aralkyl group, and R C is an optionally substituted arylcarbonyl group, and R D is an optionally substituted aryl group, and R F is an optionally substituted aryl group.

[0060] In the glycosylation reaction using compound (11), an activating agent is added to activate compound (11). 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.

[0061] Fifth Embodiment The fifth embodiment of the present invention involves reacting a compound represented by formula (1) (hereinafter also referred to as "compound (1)") 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). [ka]

[0062] <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. [ka]

[0063] In formula (1), X is —O—, —NR— or —S—.

[0064] In formula (1), R represents a hydrogen atom, an optionally substituted C 1-6 alkyl group, optionally substituted C 3-6 Cycloalkyl groups, optionally substituted C 2-6 Alkenyl group, optionally substituted C 3-6 Cycloalkenyl group, optionally substituted C 2-6 Alkynyl group, optionally substituted C 6-10 Aryl group, optionally substituted C 1-6 Alkylcarbonyl group, optionally substituted C 6-10 Arylcarbonyl group, optionally substituted C 1-6 Alkyl sulfonyl group or optionally substituted C 6-10 It is an arylsulfonyl group.

[0065] In formula (1), R 1 , R 2 , R 3 and R 4 are each independently a hydrogen atom, C 1-6 Alkyl group, C 1-6 Alkoxy group, C 3-6 Cycloalkyl groups, C 2-6 Alkenyl group, C 3-6 Cycloalkenyl group, C 2-6 Alkynyl group or C 6-10 It is an aryl group.

[0066] C 1-6 The alkyl group means an alkyl group having 1 to 6 carbon atoms. 1-6Examples of the 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.

[0067] C 1-6 An alkoxy group is a C 1-6 It is an alkyl group. 1-6 Examples of the oxyalkyl group include a methoxy group, an ethoxy group, a propoxy group, a butoxy group, and a pentoxy group.

[0068] C 3-6 The cycloalkyl group means a cycloalkyl group having 3 to 6 carbon atoms. 3-6 Examples of the cycloalkyl group include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, and a cyclohexyl group.

[0069] C 2-6 The alkenyl group means an alkenyl group having 2 to 6 carbon atoms. 2-6 Examples of 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.

[0070] C 3-6 The cycloalkenyl group means a cycloalkenyl group having 3 to 6 carbon atoms. 3-6 Examples of the cycloalkenyl group include a cyclobutenyl group, a cyclopentenyl group, and a cyclohexenyl group.

[0071] C 2-6 The alkynyl group refers to an alkynyl group having 2 to 6 carbon atoms. 2-6Alkynyl groups include, for example, ethynyl, propargyl, and 3-butyn-1-yl groups.

[0072] C 6-10 The aryl group means an aryl group having 6 to 10 carbon atoms. 6-10 Aryl groups include, for example, phenyl and naphthyl groups.

[0073] C 1-6 Alkyl group, C 3-6 Cycloalkyl groups, C 2-6 Alkenyl group, C 3-6 Cycloalkenyl group, C 2-6 Alkynyl groups and C 6-10 The aryl group may be unsubstituted or substituted. The substituents include C 1-4 Alkyl group, C 2-4 Alkenyl group, C 2-4 Alkynyl group, C 1-4 Examples of the alkyl group include an alkoxy group, an amino group, an imino group, a nitro group, an oxy group, an oxo group, a nitrile group, a mercapto group, and a halogen atom.

[0074] Examples of the compound (1) include cis-2,3-epoxybutane and 3,4-epoxyhexane.

[0075] In formula (1), R 2 and R 3 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, R 1 and R 4 is the same as the definition in equation (1). [ka]

[0076] R 2a and R 3a are R 2 and R 3 It is a divalent group expressed by replacing the hydrogen atom with a single bond.2a and R 3a are each independently C 1-6 Alkylene group, C 1-6 Oxyalkylene group, C 3-6 Cycloalkylene group, C 2-6 Alkenylene group, C 3-6 Cycloalkenylene group, C 2-6 Alkynylene group or C 6-10 It is an arylene group.

[0077] R 2a -R 3a Examples of the group represented by the formula (I) include a 2-oxapropylene group (-CH2OCH2-), a 3-oxopentylene group (-CH2CH2OCH2CH2-), a 3-oxopentylene group (-CH2CH2C(=O)CH2CH2-), and an acetonide group (-CH2-OC(CH3)2-O-CH2-).

[0078] 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.

[0079] <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 (HO) and hydrogen sulfide (HS) are excluded from the scope of compound (2). [ka]

[0080] In equation (2), R 5 is a hydrogen atom, optionally substituted C 1-6alkyl group, optionally substituted C 3-6 Cycloalkyl groups, optionally substituted C 2-6 Alkenyl group, optionally substituted C 3-6 Cycloalkenyl group, optionally substituted C 2-6 an alkynyl group or an optionally substituted C 6-10 It is an aryl group.

[0081] In equation (2), R 6 is a hydrogen atom, optionally substituted C 1-6 alkyl group, optionally substituted C 3-6 Cycloalkyl groups, optionally substituted C 2-6 Alkenyl group, optionally substituted C 3-6 Cycloalkenyl group, optionally substituted C 2-6 an alkynyl group or an optionally substituted C 6-10 It is an aryl group.

[0082] C 1-6 Examples of the 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.

[0083] C 1-6 An alkoxy group is a C 1-6 It is an alkyl group. 1-6 Examples of the oxyalkyl group include a methoxy group, an ethoxy group, a propoxy group, a butoxy group, and a pentoxy group.

[0084] C 3-6 Examples of the cycloalkyl group include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, and a cyclohexyl group.

[0085] C 2-6Examples of 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.

[0086] C 3-6 Examples of the cycloalkenyl group include a cyclobutenyl group, a cyclopentenyl group, and a cyclohexenyl group.

[0087] C 2-6 Alkynyl groups include, for example, ethynyl, propargyl, and 3-butyn-1-yl groups.

[0088] C 6-10 Aryl groups include, for example, phenyl and naphthyl groups.

[0089] C 1-6 Alkyl group, C 3-6 Cycloalkyl groups, C 2-6 Alkenyl group, C 3-6 Cycloalkenyl group, C 2-6 Alkynyl groups and C 6-10 The aryl group may be unsubstituted or substituted. The substituents include C 1-4 Alkyl group, C 2-4 Alkenyl group, C 2-4 Alkynyl group, C 6-10 Aryl group, C 1-4 Examples of the alkyl group include an alkoxy group, an amino group, an imino group, a nitro group, an oxy group, an oxo group, a nitrile group, a mercapto group, and a halogen atom.

[0090] In one embodiment, compound (2) is R 5 or R 6When compound (2) has an asymmetric center, compound (2) exists as 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 a racemic compound (2) is used, one of the optical isomers may proceed preferentially in this reaction. For example, R 5 or R 6 The stereoselectivity can be further enhanced by carrying out the asymmetric ring-opening reaction using compound (2) which has an asymmetric center at R. 5 or R 6 By removing the chiral center, a product with higher optical purity can be obtained than when the asymmetric ring-opening reaction is carried out using compound (2) which does not have an asymmetric center.

[0091] 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 ...propylamine, 2-propylamine (isopropylamine), 2-propylamine, 2-propylamine, 3-pentylamine, cyclopropylamine, cyclobutylamine, cyclopentylamine, cyclohexylamine, tert-butylamine, sec-butylamine, methylamine, ethylamine, propylamine, propylamine, propylamine, propylamine, propylamine, propylamine, propylamine, propylamine, propylamine, propylamine, propylamine, propylamine, propylamine, propylamine, propylamine, propylamine, propylamine, propylamine Examples of suitable amines include ethylamine (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.

[0092] In equation (2), R 5 and R 6may 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 the same as defined in formula (2). In formula (2a), R 5a and R 6a represents a group formed by bonding together. [ka]

[0093] R 5a and R 6a are R 5 and R 6 It is a divalent group expressed by replacing the hydrogen atom with a single bond. 5a and R 6a are optionally substituted C 1-6 Alkylene group, optionally substituted C 1-6 Oxyalkylene group, optionally substituted C 3-6 Cycloalkylene group, optionally substituted C 2-6 Alkenylene group, optionally substituted C 3-6 Cycloalkenylene group, optionally substituted C 2-6 Alkynylene group or C 6-10 It is an arylene group.

[0094] R 5a -R 6a Examples of the group represented by the formula (I) include a 2-oxapropylene group (-CH2OCH2-), a 3-oxopentylene group (-CH2CH2OCH2CH2-), and a 3-oxopentylene group (-CH2CH2C(=O)CH2CH2-).

[0095] Specific examples of the compound (2a) include pyrrolidine, piperidine, morpholine, piperazine, homopiperazine, and thiomorpholine.

[0096] C 1-6 Alkyl group, C 3-6 Cycloalkyl groups, C 2-6 Alkenyl group, C 3-6 Cycloalkenyl group, C2-6 Alkynyl groups and C 6-10 The aryl group may be unsubstituted or substituted. The substituents include C 1-4 Alkyl group, C 2-4 Alkenyl group, C 2-4 Alkynyl group, C 1-4 Examples of the alkyl group include an alkoxy group, an amino group, an imino group, a nitro group, a hydroxy group, an oxo group, a nitrile group, a mercapto group, and a halogen atom. 1-4 Alkoxy groups include, for example, methoxy, ethoxy, propyloxy, and butoxy groups.

[0097] The amount of compound (2) can be any amount taking into consideration economic efficiency and recoverability, and 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).

[0098] <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 is the same as the definition above. [ka]

[0099] 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). [ka]

[0100] Specific examples of compound (3) include 1,2-diol (when X and Y are both -O-), 1,2-aminoalcohol (when X is -O- and Y is -NH-, or when X is -NH- and Y is -O-), 1,2-diamine (when X and Y are both -NH-), 1,2-mercaptoalcohol (when X is -O- and Y is -S-), and 1,2-mercaptoamine (when X is -NH- and Y is -S-).

[0101] 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). [ka] In the formula, R 1 , R 2 , R 3 and R 4 are each independently a hydrogen atom, an optionally substituted C 1-6 alkyl group, optionally substituted C 3-6 Cycloalkyl groups, optionally substituted C 2-6 Alkenyl group, optionally substituted C 3-6 Cycloalkenyl group, optionally substituted C 2-6 an alkynyl group or an optionally substituted C 6-10 is an aryl group, and R 2 and R 3may be bonded to each other to form a cyclic structure. For example, by mixing trans-2-chlorohexanol with cyclopropylamine instead of 7-oxabicyclo[4.1.0]heptane in the presence of the above-mentioned polysaccharide, crude pectic polysaccharide, or galactan, trans-2-chlorohexanol is converted to 7-oxabicyclo[4.1.0]heptane, which is then converted to 2-cyclopropylaminocyclohexanol by a catalytic reaction. For reaction conditions, see the first embodiment.

[0102] <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).

[0103] The asymmetric ring-opening reaction according to the embodiment of the present invention may be carried out in a solvent. When the reaction is carried out in a solvent, organic solvents well known in organic synthetic chemistry and water can generally be used as long as the solvent does 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 a mixture of two or more solvents is used, it is preferable to use a mixture of an organic solvent and water. From the viewpoints of recovery, safety, and economy, it is particularly preferable to use a mixture of toluene or heptane with water.

[0104] The amount of the solvent that can be used in the asymmetric ring-opening reaction, whether it is a single solvent or a mixed solvent, can be determined in consideration of economic efficiency. The amount of such a 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.

[0105] 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. Within such a range, the conversion rate of the reaction and the optical purity of the product are further improved.

[0106] The reaction temperature is preferably -20°C to 100°C, and from the viewpoint of improving catalytic activity, particularly preferably 30°C to 70°C or 30°C to 50°C. After completion of the reaction, the catalyst can be filtered off to obtain the corresponding compound (3). The catalyst recovered by filtration can be reused when carrying out the asymmetric ring-opening reaction of the present invention.

[0107] The reaction can be carried out for a time period that provides any desired conversion rate, taking economic 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.

[0108] After the reaction is complete, the catalyst is filtered off to obtain compound (3). The obtained compound (3) can be easily purified by standard methods such as crystallization and distillation.

[0109] The solvent that can be used for crystallization is not particularly limited as long as it is a solvent that is normally used in organic synthetic chemistry. Examples of solvents that can be used 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.

[0110] The amount of the solvent, whether a single solvent or a mixed solvent, can be set in consideration of economic efficiency, 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) in terms of volume ratio.

[0111] <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 synthesis. Examples of such acids 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.

[0112] In order to increase the optical purity of compound (3), it may be possible to form a salt with an optically active acid, such as tartaric acid, malic acid, mandelic acid, or phenylglycine, and the acid may be substituted.

[0113] As the solvent for forming the salt, a solvent commonly used in organic synthetic chemistry can be used, taking into consideration economic efficiency, recoverability, etc. Examples of the solvent 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.

[0114] The amount of the solvent used to form the salt can be determined economically, whether it is a single solvent or a mixed solvent, and 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.

[0115] 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.

[0116] <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.

[0117] (-)-6-[3-[3-cyclopropyl-3-[(1R,2R)-2-hydroxycyclohexyl]ureido]propoxy]-2(1H)-quinolinone is a substance that has dual 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 cerebral and peripheral vascularity increasing effects. 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.

[0118] 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 the known method is the method described in Patent Document 4.

[0119] When reacting compound A and compound B, as shown in the following formula (Eq.1), compound A can be reacted with a carbonylation reagent in advance, and then compound B can be reacted. After reacting compound A with a carbonylation reagent, the reaction product may be purified. [ka] [In the formula, Z represents a residue of a carbonylation reagent.]

[0120] Step (i) can be carried out without a solvent 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.

[0121] Examples of the carbonylation reagent include chloroformates such as phenyl chloroformate, carbonates such as diethyl carbonate, carbonyldiimidazole, phosgene, and triphosgene.

[0122] The step (i) is usually carried out at a temperature of from -20 to 150°C, preferably from -20 to 100°C.

[0123] Step (i) can be carried out in the presence or absence of a basic compound. Examples of the basic compound 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.

[0124] In step (i), an additive may be further used to make the reaction proceed more efficiently. Examples of such an additive include potassium iodide, sodium iodide, imidazole, 4-dimethylaminopyridine, and 4-pyrrolidinopyridine.

[0125] Step (ii) can be carried out without a solvent or in a solvent. The solvents usable in step (ii) include those listed in step (i).

[0126] Step (ii) is usually carried out at a temperature of -20 to 150°C, preferably -20 to 100°C.

[0127] Step (ii) can be carried out in the presence or absence of a basic compound. The basic compounds usable in step (ii) include the basic compounds listed in step (i).

[0128] Alternatively, as shown in the following formula (Eq.2), compound B may be reacted with a carbonylating reagent in advance, followed by reaction with compound A. In this case, each step can be carried out in the same manner as in formula (Eq.1). [ka] [In the formula, Z represents a residue of a carbonylation reagent.]

[0129] Compound B used in the present invention may be a protected compound B. The protected compound B may be one in which the 1-position of 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.

[0130] Alternatively, 6-(3-aminopropoxy)-quinoline substituted at the 2-position can also be used as the protected compound B. Examples of the substituent of 6-(3-aminopropoxy)-quinoline substituted at the 2-position include a halogen atom such as a fluorine atom, a chlorine atom, or a bromine atom; an alkoxy group such as a methoxy group or a methoxymethoxy group; an arylalkyloxy group such as a benzyloxy group; an acyloxy group such as an acetoxy group or a pivaloyloxy group; and a silyloxy group such as a triethylsilyloxy group.

[0131] When a protected compound B is used in the reaction, it can be deprotected by a known method after the reaction to convert it to (-)-6-[3-[3-cyclopropyl-3-[(1R,2R)-2-hydroxycyclohexyl]ureido]propoxy]-2(1H)-quinolinone. As the conditions for such deprotection, the method described in Protective Groups in Organic Synthesis, John Wiley and Sons (1980) can be used, and examples thereof include acidic conditions, alkaline conditions, and hydrogenation.

[0132] 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.

[0133] 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: [ka]

[0134] In the above formula, R Xis an organic group, for example, R X may be substituted C 1-6 alkyl group, optionally substituted C 3-6 Cycloalkyl groups, optionally substituted C 2-6 Alkenyl group, optionally substituted C 3-6 Cycloalkenyl group, optionally substituted C 2-6 Alkynyl group, optionally substituted C 6-10 Aryl group, optionally substituted C 1-6 Alkylcarbonyl group, optionally substituted C 6-10 Arylcarbonyl group, optionally substituted C 1-6 Alkyl sulfonyl group or optionally substituted C 6-10 The arylsulfonyl group is defined as an arylsulfonyl group. The definitions of each group can be found in the above descriptions.

[0135] 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 from the polysaccharide according to the first embodiment by reductive amination, R X is the amine (R X -NH2) chemical structure. For example, R X When is a p-ethoxycarbonylphenyl group or a tert-butoxycarbonylphenyl group, the amines used are ethyl p-aminobenzoate (ABEE) and tert-butyl p-aminobenzoate (ABtB), respectively. [ka]

[0136] The amino sugar according to this embodiment also exhibits catalytic activity and stereoselectivity in asymmetric ring-opening reactions with nucleophiles, similar to the polysaccharides according to the first to third embodiments. Furthermore, the amino sugar according to the fifth embodiment is ABEE- or ABtB-conjugated, and therefore can also be used as a label. [Example]

[0137] The analytical methods used in the following Examples and Comparative Examples will be explained below.

[0138] (1) Catalytic activity measurement method I (100 mg catalyst scale) A measurement sample (200 mg) was weighed into a 15 mm inner diameter flat-bottom screw-cap test tube, 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 to 10 times with toluene, dehydrated using anhydrous magnesium sulfate or anhydrous sodium sulfate, and subjected to GC analysis.

[0139] GC analysis was carried out under the following conditions: Equipment: GC-2014 (Shimadzu Corporation) Column: BETA DEXTM325 (30m x 0.25mm x 0.25μm) Flow rate: 94kPa (inlet pressure) Split ratio: 1:30 Carrier gas: Helium Column temperature: 120℃ Vaporization chamber temperature: 230℃ Detector: FID (temperature 300℃)

[0140] 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. 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).

[0141] The stereoselectivity of the catalyst was expressed as the optical purity (%ee) of the product and calculated as follows:

[0142] 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} × 100

[0143] (2) Catalytic activity measurement method II (when measuring only the conversion rate) 5 mg of polysaccharide sample was weighed into a 2 mL glass vessel, 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 1000 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."

[0144] (3) Catalytic activity measurement method III (5 mg catalyst amount) Five mg of polysaccharide sample was weighed into a 2 mL glass vessel, 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 stir 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."

[0145] (4) Catalytic activity measurement method IV (0.1 mg catalyst amount) 0.1 mg of polysaccharide sample was weighed into a 2 mL glass vessel, and 100 μL of a toluene solution of the substrate (1.0 M 1,2-epoxycyclohexane, 1.5 M cyclopropylamine) and 0.2 μL of saturated saline were added. The reaction was carried out at 37°C and 1000 rpm for 20 hours using a stir bar. After the reaction, catalytic activity (mU / mg) and stereoselectivity (%ee) were measured by GC analysis as described in "Catalytic Activity Measurement Method I."

[0146] (5) Catalytic activity measurement method V (5~10mg catalyst amount) 5–10 mg of polysaccharide sample was weighed into a 2 mL glass vessel, 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 1000 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).

[0147] (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 above 10 kDa, a TOSOH TSKgel G3000PW (7.5 mm I.D. x 30 cm, Tosoh Corporation) was used, and for samples below 10 kDa, 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 a RID detector.

[0148] (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 min, and RID detector. For samples above 10 kDa, 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 in conjunction with a column. 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 consisting of 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.

[0149] (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 (monoglycosylated) lyophilized material was dissolved again in 50 μL of purified water to obtain the monoglycosylated 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 monoglycosylated 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, and impurities were extracted into the organic layer. The aqueous layer was analyzed by HPLC. The column used was a TSKGel ODS120H (manufactured by Tosoh Corporation, trade name), and the mobile phase was a mixed solvent of 50 mM borate buffer (pH 9.0) / acetonitrile (volume ratio 91:9). The analysis was performed under conditions of a flow rate of 0.8 mL / min, a column temperature of 35°C, and a UV detection wavelength of 310 nm.

[0150] (8) MALDI-TOFMS analysis method The instrument used was a JMS-S3000 Spiral TOF-plus (manufactured by JEOL) and 2,5-dihydroxybenzoic acid 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 above 3000.

[0151] Example A1: Preparation of crude pectic polysaccharides from plants (1) Preparation of degreased samples 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 then covered with aluminum foil and vigorously stirred for 1 hour using a stir bar. 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 above mixture. The collected plant matter and 300 mL of the above mixture were added to the flask, and after stirring again for 1 hour using a stir bar, the plant matter was collected by filtration. This process was repeated two more times (for a total of three times) to obtain a defatted sample.

[0152] (2) Preparation of soluble polysaccharide-removed samples 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 then 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.

[0153] (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 at 121°C for 3 hours. The mixture was then transferred to a 500 mL centrifuge bottle and centrifuged at 17,800 × g for 10 minutes at 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 stir bar. The precipitate was collected by centrifugation at 17,800 × g for 10 minutes at 4°C, dissolved in RO water, and the supernatant was collected and lyophilized to obtain a crude pectic polysaccharide sample.

[0154] (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. Vigorous stirring was performed for 4 hours using a stir bar. The sample was transferred to a 500 mL centrifuge bottle and centrifuged (17,800 × g, 10 minutes, 4°C). The resulting supernatant was then subjected to ethanol precipitation and freeze-drying in the same manner as in (3) above.

[0155] (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 added sequentially to a 1000 mL Erlenmeyer flask. The Erlenmeyer flask was covered with aluminum foil and heated in a 90°C water bath. The mixture was vigorously stirred for 1 hour using a stir bar. Centrifugation, ethanol precipitation, and freeze-drying were carried out in the same manner as in (3) above.

[0156] (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. The mixture was stirred vigorously for 1 hour using a stirrer. Centrifugation, ethanol precipitation, and freeze-drying were carried out in the same manner as in (3) above.

[0157] 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 listed in Tables 1 and 2. In the tables, "A" indicates that a defatted sample was used, and "B" indicates that crude pectic polysaccharide prepared by the acetic acid method was used. [Table 1] TIFF0007723394000033.tif136149

[0158] [Table 2] TIFF0007723394000035.tif226149TIFF0007723394000036.tif226149TIFF0007723394000037.tif124149Abbreviations used 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., R T: 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: Japan Garlic Co., Ltd., TS: Japan Beet Sugar Co., Ltd., CP: Commpro Co., Ltd., KD: Kurodaya Co., Ltd., KW: Kawadaen, HO: Honoka Ogre Farm, HE: HERBS & CROPS, SE: Eno Brostup SA, 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 in A is the amount of defatted sample prepared from 50 g of plant matter, and the yield in B is the weight of crude pectic polysaccharides prepared from 30 g of defatted sample (15 g for seaweed).

[0159] 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, along with the yield, are shown 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. [Table 3]

[0160] By extracting crude pectic polysaccharides, it was found that sufficient catalytic activity and stereoselectivity of 30% ee or more were exhibited even from plants of the families 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, which are not described in Patent Documents 7, 8, and Non-Patent Document 11. Furthermore, stereoselectivity of 30% ee or more was also exhibited from plants of the families Poaceae, Cucurbitaceae, Solanaceae, Rosaceae, Theaceae, and Amaryllidaceae, which had previously shown low stereoselectivity. Furthermore, catalytic activity (specific activity per unit weight) was improved by up to 17-fold.

[0161] Example A2: Preparation of LP-LMW from plant material (water-soluble soybean polysaccharides) 7.5 g of water-soluble soybean polysaccharide (trade name: SOYAFIBE S-DN, 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 (Amano Enzyme Inc.) in 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 autoclave, and the mixture was shaken at 37°C and 80 rpm for 20 hours. 40 mg of Proteinase K (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 min, 4 °C). Acetone (0.6 times the volume of the pH-adjusted solution) was added to the resulting supernatant and stirred for 10 min using a stir bar. The resulting precipitate was then centrifuged (10,000 × g, 10 min, 4 °C), dissolved in purified water, and sterilized by autoclaving (121 °C, 15 min). The mixture was centrifuged (15,000 × g, 10 min, 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 solution in the dialysis tube was lyophilized, yielding 2.2 g of the LP-LMW fraction.

[0162] The catalytic activity, stereoselectivity, and average molecular weight of the main peak of water-soluble soybean polysaccharide (product name: SOYAFIVE 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. Catalytic activity and stereoselectivity were measured using "Catalytic Activity Measurement Method III," and average molecular weight was measured using "Gel Filtration HPLC Analysis Method II." [Table 4] [Table 5]

[0163] Example A3: Degradation of polysaccharides by rhamnogalacturonate lyase (RGL) (1) Purification of "enzyme that decomposes water-soluble soybean polysaccharides" (Lip-RGL) from commercially available enzymes 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.

[0164] The solution was applied to an FPLC system (AKTA explorer 10S, GE Healthcare) equipped with a Mono Q 10-100 GL column (8.0 mL, GE Healthcare), and eluted with 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 eluted with 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 and a single band on SDS-PAGE were collected, desalted, and purified water-soluble soybean polysaccharide-degrading enzyme (Lip-RGL).

[0165] 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.

[0166] (2) Comparison of enzyme activity 25 mg of water-soluble soybean polysaccharide (trade name: SOYAFIBE S-DN, 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, Amano Enzyme Inc.) 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 the filtrate. The molecular weight distribution of the resulting filtrate was evaluated using a TSKgel G5000PWXL column (Tosoh Corporation) under the conditions of "Gel Filtration HPLC Analysis I." Comparison of the chromatograms showed that degradation products with similar molecular weights were produced in both reactions, demonstrating that Lip-RGL can degrade water-soluble soybean polysaccharides.

[0167] Furthermore, when the protein concentrations of both enzyme solutions were compared, 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.

[0168] (3) Obtaining the LipRGL gene and introducing it into E. coli The 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.

[0169] 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 therefore compared the obtained amino acid sequence information with entries from the genus Aspergillus (taxid: 5052) in the NCBI protein database, extracted five enzymes (glucoamylase, cellobiohydrolase, rhamnogalacturonate lyase, endo-1,3-β-D-glucanase, and β-mannase) as candidates, and presumed that this enzyme was rhamnogalacturonate lyase because it digests water-soluble soybean polysaccharides with a rhamnogalacturonan structure.

[0170] (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) is publicly available 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 peptide of LipRGL.

[0171] 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. Based on the obtained gene, forward (SEQ ID NO: 3) and reverse (SEQ ID NO: 4) PCR primers were prepared. The gene was amplified by PCR using these primers and the artificially synthesized 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 extracts of each transformant were able to produce the catalyst (AkRGL-LMW) from SoyaFive S-DN, similar to lipase (Lipase A "Amano" 6, Amano Enzyme Co., Ltd.).

[0172] (5) Preparation of recombinant RGL (Ak-RGL) E. 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. 1 mM IPTG was added, and the culture was further cultured at 37°C for 12 hours. 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 beads using a Multi-Beads Shocker (Yasui Kikai Co., Ltd.). Insoluble material was removed by centrifugation (12000 × g, 15 minutes, 4°C) to obtain an extracellular extract.

[0173] The extracellular extract was applied to a Ni Sepharose 6 Fast Flow column (GE Healthcare Life Sciences, 3 x 8.5 cm) and washed with 10 mM potassium phosphate buffer (pH 8.0) containing 0.1 M sodium chloride and 10 mM imidazole. The column was then eluted by successively increasing the imidazole concentration in the same buffer up to 300 mM. Enzyme-active fractions were pooled, desalted, and loaded onto 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, the column was eluted by successively increasing the imidazole concentration in 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.

[0174] (6) Digestion of SoyaFive 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 lipase (Lipase A "Amano" 6, Amano Enzyme Inc.) solution, purified LipRGL solution, or purified AkRGL solution was added. After heating at 37°C for 2 hours, the digestion solution was lyophilized to obtain the digest. The catalytic activity and stereoselectivity of the resulting digest were measured according to "Catalytic Activity Measurement Method II." [Table 6]

[0175] As shown in Table 6, both the digests prepared by purified LipRGL and purified AkRGL had catalytic activity and stereoselectivity equivalent to those of SOYAFIBE S-DN. The catalytic activity of the digest prepared by purified AkRGL (AkRGL-LMW) was 25.1 mU / mg.

[0176] Example A4: Decomposition with alkali (1) Preparation by sodium hydroxide and ethanol fractionation To an aqueous solution (2.5 L) of sodium hydroxide (200 g) and sodium borohydride (7.6 g), 100 g of water-soluble soybean polysaccharide (trade name: SOYAFIBE S-DN, manufactured by Fuji Oil Co., Ltd.) was added, placed in a 3 L four-neck flask, and heated with stirring in an oil bath at 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.

[0177] 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 × 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 × g, 20 minutes). The supernatant was used in the next step. The resulting precipitate was suspended in purified water and placed in a dialysis tube with a MWCO of 3500 (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).

[0178] Ethanol (0.3 volume of ethanol) was added dropwise to the supernatant obtained in the previous step while stirring. The solution after addition was left to stand in a refrigerator for 3 days, and the resulting precipitate was collected by centrifugation (12,000 × 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 × 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, and then placed in a 3500 MWCO 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).

[0179] Ethanol (0.3 volume of X) was added dropwise to the supernatant obtained in the previous step while stirring. The solution after addition was left to stand in a refrigerator for 1 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 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 of each fraction are shown in Table 8.

[0180] [Table 7] [Table 8]

[0181] (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.

[0182] Acetone (0.5 volume of X) was added dropwise to the neutralized hydrolysis solution while stirring. The resulting precipitate was collected by centrifugation (12,000 × 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).

[0183] 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 × 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).

[0184] Acetone (0.3 volume of X) was added dropwise to the supernatant obtained in the previous step while stirring. 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 freeze-dried to obtain 2.5 g of a polysaccharide fraction (AL-LMW Acetone 0.8 / 1.1).

[0185] The catalytic activity, stereoselectivity, and average molecular weight of each fraction were measured, and the results are shown in Table 9. 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." The results of monosaccharide analysis of each fraction are shown in Table 10.

[0186] [Table 9] [Table 10]

[0187] (3) Temperature considerations Four grams of water-soluble soybean polysaccharide (product name: SOYAFIBE S-DN, Fuji Oil Co., Ltd.) was added to 200 mL of an aqueous solution of sodium hydroxide (16 g) and sodium borohydride (0.30 g). Thirty mL of the water-soluble soybean polysaccharide solution was placed in five 30 mm 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 test tubes, fitted with silicone stoppers, and heated in an autoclave at 121 °C for 5 hours. 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 tubes were left 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 10 mM acetic acid solution. The dialyzed solution was centrifuged (15,000 × g, 10 minutes), and the supernatant was freeze-dried to obtain the polysaccharide fraction (AL-LMW).

[0188] The hydrolysis time, yield, catalytic activity, stereoselectivity, average molecular weight, and visual color of the hydrolyzed solution at the end of hydrolysis for 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. [Table 11]

[0189] (4) Consideration of alkali concentration Water-soluble soybean polysaccharides (trade name: SOYAFIBE S-DN, 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, or 3.6 g (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 to 70 °C in a heat block 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. After adjusting the pH, ethanol (3 times the volume of X) was added dropwise to the solution and 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 the polysaccharide fraction (AL-LMW).

[0190] The decomposition time, yield, catalytic activity, stereoselectivity, peak molecular weight, and color of the hydrolyzate at the end of decomposition for each alkali concentration are shown in Table 12. 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.

[0191] [Table 12]

[0192] (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.

[0193] 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." [Table 13]

[0194] Example A5: Preparation of AL-LMW from "Okara" Five grams of okara powder (trade name: Okara Powder, manufactured by Acrosia Corporation) was suspended in 250 mL of an aqueous solution containing 20 g of sodium hydroxide and 0.38 g of sodium borohydride. The suspension was placed in a 300 mL four-neck flask and heated in an oil bath at 70°C for 100 hours with stirring. The pH of the reaction solution was adjusted to 6 with acetic acid, and the hydrolyzed solution was centrifuged (12,000 × g, 15 minutes). The volume of the resulting supernatant (X) was measured. Ethanol (0.5 volume of X) was added dropwise to the supernatant while stirring, and the resulting precipitate was removed by centrifugation (12,000 × g, 15 minutes). Ethanol (0.3 volume of X) was added dropwise to the supernatant of this centrifuged solution, and the resulting precipitate was removed by centrifugation (12,000 × g, 15 minutes).

[0195] 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 × 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 × 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 × g, 15 minutes). The resulting precipitates were each dissolved in purified water and stored in a refrigerator for 1 to 3 days. The precipitates that formed during refrigeration were centrifuged and separated into precipitate (PPT) and 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 dialyzed solution was lyophilized to obtain three fractions (EtOH 0.8 / 1.1, EtOH 1.1 / 1.4, and EtOH 1.4 / 2.0) of PPT and SUP, respectively.

[0196] 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. [Table 14] [Table 15]

[0197] Example A6: Enzymatic Degradation of LP-LMW (1) Enzyme selection 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 mixture, and the mixture was reacted at 37°C and 1000 rpm for an additional 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 amounts of enzymes A, B, or E added. [Table 16]

[0198] The results of time-dependent analysis under gel filtration HPLC analysis conditions I using 0.46 mU / mL of enzyme A are shown in Figure 3. Figure 3 shows chromatograms of untreated (LP-LMW) and after 0.5, 1, 2, 4, 6, or 24 hours of treatment. Treatment with endo-β1,4-galactanases from different species resulted in degradation of LP-LMW to a molecular weight of 3,000 or less, suggesting that the main component of LP-LMW is β1,4-linked galactose.

[0199] (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 (product name: DEAE TOYOPEARL 650, 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 fractions. The resulting fractions were measured for 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.

[0200] 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. [Table 17] [Table 18]

[0201] (3) Partial degradation of the D1 fraction of LMW (LP-LMW-D1) by enzyme treatment LP-LMW-D1 (343 mg) was weighed into a 200 mL Erlenmeyer flask and dissolved in 32 mL of purified water and 8 mL of 0.5 M MES buffer (pH 6.0) (final concentration: 10 mg / mL LMW, 100 mM MES buffer (pH 6.0)). Enzyme A (4 μL, final concentration: 1.8 mU / mL) 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 reaction was continued 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-D1 deg (280 mg). The catalytic activity and stereoselectivity of the obtained fractions were measured according to "Catalytic Activity Measurement Method III," and the peak molecular weight was measured according to "Gel Filtration HPLC Analysis Method II," and the results are shown in Table 18 together with the yield.

[0202] (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. 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 fractions of 199 mg and 37.2 mg, respectively.

[0203] 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.

[0204] [Table 19] [Table 20]

[0205] (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.

[0206] The obtained fraction was analyzed according to "Gel filtration HPLC analysis method II" and found to be 5 × 10 3 Super, 4~5×10 3 , 3 to 4 × 10 3 , 2-3×10 3 , 2 × 10 3 The solution was collected into five fractions of less than 1000 mg each and freeze-dried 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.

[0207] The catalytic activity, stereoselectivity, average molecular weight, and monosaccharide analysis results for 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. [Table 21]

[0208] (6) Degradation of AL-LMW by galactanase AL-LMW was purified using an anion exchange resin in the same manner as 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 the 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" to find 0.2 mol% rhamnose, 0.3 mol% arabinose, 96.4% galactose, and 0.1 mol% galacturonic acid; no other monosaccharides were detected. [Table 22]

[0209] 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. 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." [Table 23]

[0210] (7) Relationship between molecular weight and catalytic activity, and between molecular weight and stereoselectivity The relationship between molecular weight, catalytic activity, and stereoselectivity for the fractions obtained by digesting or purifying LP-LMW and AL-LMW is summarized in Figure 4. 3 ~3×10 3 The stereoselectivity of the catalyst decreased rapidly between the molecular weight of 2.5 × 10 3 ~6×10 3 A tendency for the performance to improve with molecular weight was observed.

[0211] Example A7: Hydrolysis of polysaccharides with acid (1) Examination of acidic conditions Five grams 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.

[0212] 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 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 1 day. The resulting precipitate was collected by centrifugation (15,000 × 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.

[0213] The AL-LMW EtOH 0.8 / 1.1-PPT solution was adjusted to pH 3 with dilute sulfuric acid, and 30 mL of the solution was placed in test tubes and heated at internal temperatures of 70°C, 80°C, or 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 and stored in a refrigerator for 1 day. The resulting precipitate was collected by centrifugation (15,000 × 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.

[0214] The AL-LMW EtOH 0.8 / 1.1-PPT solution was adjusted to pH 3 with dilute sulfuric acid, and 7 mL of the solution was transferred to a test tube, fitted with a silicone stopper, and heated to 121°C in an autoclave. After hydrolysis had progressed, 4.75 mL of a sample taken from the test site was added 0.25 mL of 1 M sodium acetate solution (pH 5) and 15 mL of ethanol, successively, and stored in a refrigerator for 1 day. The resulting precipitate was collected by centrifugation (15,000 × 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.

[0215] Table 24 shows the hydrolysis time, yield of the resulting low-molecular-weight polysaccharide fraction, catalytic activity, stereoselectivity, and average molecular weight of the resulting hydrolysis products under each hydrolysis condition. 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 may exceed 100% due to the inclusion of inorganic salts. Acid hydrolysis was confirmed to proceed at temperatures ranging from 70 to 121°C and pH values ranging from 2 to 4. [Table 24]

[0216] (2) Hydrolysis with sulfuric acid The AL-LMWEtOH0.8 / 1.1 PPT fraction (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.

[0217] Thereafter, the pH was adjusted to 6 to 7 with 1 M sodium hydroxide, and insoluble matter was removed by centrifugation (12,000×g, 20 minutes) to obtain a neutralized hydrolyzed solution, and its volume (X) was measured.

[0218] Ethanol (same amount as X) was added dropwise to the neutralized hydrolyzed solution 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 1,000, 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).

[0219] 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 × g, 20 minutes). (The separated supernatant 2 was used in the next step.) The resulting precipitate was suspended in purified water, placed in a dialysis tube with a MWCO of 1,000, and dialyzed against 10 mM acetic acid solution as the external solution. The dialyzed solution was freeze-dried to obtain 6.2 g of acid-treated low molecular weight polysaccharide fraction (AS-LLMW EtOH 1.0 / 2.0).

[0220] Ethanol (same amount as X) was added dropwise to Supernatant 2 and stirred. The resulting precipitate was collected by centrifugation (12,000 × g, 20 minutes) (the separated Supernatant 3 was used in the next step). The resulting precipitate was suspended in purified water, placed in a dialysis tube with a MWCO of 1,000, and dialyzed against 10 mM acetic acid solution as the external solution. The dialyzed solution was freeze-dried to obtain 0.96 g of a low molecular weight polysaccharide fraction (AS-LLMW EtOH 2.0 / 3.0).

[0221] 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 the 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.

[0222] [Table 25] [Table 26]

[0223] (3) Hydrolysis using 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, stirred to dissolve, and then placed in a 500 mL four-neck flask. Regenerated Amberlyst 15R (equivalent to 10.8 g of dry) was placed in the flask, a reflux condenser was attached, and the flask 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.

[0224] The pH was then adjusted to 6-7 with 1 M aqueous sodium hydroxide, and the solution was centrifuged (12,000 × g, 20 min) 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 × g, 20 min). Ethanol (0.4 volume of X) was added dropwise to the supernatant 1 while stirring. The resulting precipitate was collected by centrifugation (12,000 × g, 20 min). (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. 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).

[0225] 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 × g, 20 minutes). (Separated supernatant 3 was used in the next step.) The resulting precipitate was suspended in purified water, placed in a dialysis tube with a MWCO of 1,000, and dialyzed against 10 mM acetic acid solution as the external solution. The dialyzed solution was lyophilized to obtain 2.0 g of a low molecular weight polysaccharide fraction (AM-LLMW EtOH 1.4 / 2.0).

[0226] Ethanol (the same amount as X) was added dropwise to the supernatant 3 while stirring. 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 1,000, and dialyzed against 10 mM acetic acid solution as the external solution. The dialyzed solution was freeze-dried to obtain 0.52 g of a low molecular weight polysaccharide fraction (AM-LLMW EtOH 2.0 / 3.0).

[0227] 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 27]

[0228] 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. [Table 28]

[0229] (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 and stirred to dissolve. 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 volume as X) was added dropwise to the neutralized hydrolysis solution while stirring. 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 10 mM acetic acid 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).

[0230] Ethanol (0.6 volume of X) was added dropwise to the supernatant 1 while stirring. The resulting precipitate was collected by centrifugation (15,000 × g, 10 minutes). (The separated supernatant 2 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 0.15 g of the acid-treated low molecular weight fraction (AA-LLMW EtOH 1.4 / 2.0).

[0231] Ethanol (the same amount as X) was added dropwise to the supernatant 2 while stirring. 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 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).

[0232] 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." [Table 29]

[0233] (5) Purification using 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) with purified water at a flow rate of approximately 8 mL / min, and fractionation was performed with a 16 mL fraction volume. The sugar-rich fraction was 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 the DEAE-unadsorbed fraction (AM-LLMW EtOH 1.4 / 2.0 D1(FT)) (1.87 g, 71% yield).

[0234] 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 3Furthermore, when monosaccharide composition analysis was performed according to the "monosaccharide composition analysis method," this fraction was found to consist of 0.1% fructose, 3.1% arabinose, and 96.8% galactose, with no other sugars detected.

[0235] (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 was applied to a BioGel P10 column (Bio-Rad Laboratories, 300 mL column, 3 cm internal diameter x 54 cm). After the sample was adsorbed, 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.

[0236] The obtained fractions were analyzed by "Gel filtration HPLC analysis II" to measure the molecular weight and relative sugar content. 3 Super, 3.5~4.5×10 3 , 3.5×10 3 After collecting the fractions less than 1000 kJ / ml, the fractions corresponding to three column volumes were combined by gel filtration chromatography and lyophilized to obtain fractions BG1 to BG3.

[0237] The catalytic activity, stereoselectivity, and average molecular weight of each fraction are shown in Table 30. 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."

[0238] [Table 30]

[0239] 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, and after stirring again for 1 hour using a stirrer, the plant matter was recovered by filtration. This process was repeated twice (for a total of 3 times), yielding 45 g of a powdered defatted sample.

[0240] Four grams 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 × 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 × g, 15 minutes, 4°C) to obtain a supernatant, and its volume (X) was measured. Ethanol (twice the amount 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).

[0241] (2) Purification of pectic polysaccharides derived from carrots 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 on an aluminum block and stirred at an internal temperature of 70°C for 40 hours. The solution was neutralized to pH 6 with acetic acid to obtain a neutralized hydrolysis solution, 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 × g, 10 minutes). Ethanol (0.6 volume of X) was added dropwise to the supernatant while stirring, and the solution was allowed to stand 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).

[0242] 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." [Table 31]

[0243] 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 fraction (AL-LMW EtOH 0.5 / 1.1), indicating improved stereoselectivity.

[0244] (3) Preparation of polysaccharide fractions and low molecular weight polysaccharide fractions from processed plant products (powdered carrots) 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). The suspension was 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). Ethanol (0.6 volume of X) was added dropwise to the resulting supernatant while stirring, and the mixture was allowed to stand 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 3500 MWCO dialysis tube. The external solution was dialyzed against 10 mM acetic acid solution. The dialyzed solution was freeze-dried to obtain 0.28 g of a polysaccharide fraction (AL-LMW EtOH 0.5 / 1.1).

[0245] 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 suspension was placed in a test tube and stirred for 96 hours while heating with an aluminum block to maintain an internal temperature of 90°C. The volume of the hydrolyzed solution was measured, and 0.053 times the volume of 1 M sodium acetate (pH 5) was added, followed by stirring (final concentration 50 mM) and centrifugation (15,000 × g, 15 minutes) to obtain a neutralized hydrolyzed solution (supernatant), and its volume (X) was measured.

[0246] 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 × g, 20 minutes). Ethanol (0.5 volume of X) was added dropwise to the resulting supernatant while stirring. This procedure was repeated twice (total of 3 times). The resulting precipitate was removed by centrifugation (15,000 × g, 20 minutes). Ethanol (the same volume as X) was added dropwise to the resulting supernatant while stirring. After leaving the solution in a refrigerator for one day, the resulting precipitate was collected by centrifugation (15,000 × g, 15 minutes). The resulting precipitate was dissolved in 1 mL of purified water and centrifuged (15,000 × 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 acid-decomposed low molecular weight polysaccharide fraction (AS-LLMW EtOH 1.0 / 2.0 DEAE-FT).

[0247] 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." [Table 32]

[0248] Compared with the raw ginseng 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 ginseng 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.

[0249] Example A9: Preparation of low molecular weight polysaccharide fractions directly from water-soluble soybean polysaccharides (1) Fractionation of water-soluble soybean polysaccharides using an anion exchange column SOYAFIBE S-DN (Fuji Oil Co., Ltd., 4 g) was dissolved in purified water (40 mL), autoclaved, and 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 in 16 mL fractions. 1 μL of the resulting fractions were applied to a TLC column, and the TLC was immersed in 10% sulfuric acid solution and heated to identify sugar-containing fractions. The fractions eluted with 10 mM and 100 mM sodium bicarbonate aqueous solutions are designated SFD1 and SFD2, respectively, and the fractions eluted with 100 mM and 300 mM sodium carbonate aqueous solutions are designated SFD3 and SFD4, respectively. Each fraction was concentrated using an evaporator, then transferred to a dialysis tube with a MWCO of 12,000 to 14,000 and dialyzed in purified water. The dialyzed solution was lyophilized, and the catalytic activity of SFD1 to SFD4 was measured according to "Catalytic Activity Measurement Method IV." The results are shown in Table 33.

[0250] [Table 33]

[0251] (2) Partial degradation of fraction SFD2 / 3 by enzyme treatment The mixture of SFD2 and SFD3 obtained above is referred to as "SFD2 / 3." A 100 mL solution containing 20 mg of SFD2 / 3 in 50 mM sodium acetate buffer (pH 5.0) 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 until the total volume reached 100 mL. Ethanol (150 mL) was added dropwise while stirring, and the mixture was stirred for another 10 minutes. After centrifugation (12,000 × g, 10 minutes, 4°C), the precipitate (SFD2 / 3degE1) and the supernatant (SFD2 / 3degE2) were obtained. 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. Then, the resulting solution was freeze-dried to obtain powders of SFD2 / 3degE1 and SFD2 / 3degE2. The yields were 79% and 12%, respectively.

[0252] (3) Fractionation of SFD2 / 3degE2 by 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 applied at a flow rate of approximately 3 mL / min using a peristaltic pump, and fractionation was performed in 10 mL fractions. Of the sugar-containing fractions, the fraction eluted with purified water was designated SFD2 / 3degE2D1 (the first fraction was designated SFD2 / 3degE2D1-1, and the second fraction 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 MWCO 1000, dialyzed against purified water, and freeze-dried.

[0253] 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. [Table 34]

[0254] (4) Fractionation of SFD2 / 3degE2D1 by gel filtration column A 300 mg / mL SFD2 / 3degE2D1 aqueous solution (1 mL) was applied to an open column (BioGel P10, Bio-Rad Laboratories, column volume (CV) 300 mL) equilibrated with 50 mM acetic acid solution. After the sample was adsorbed, the 50 mM acetic acid solution was passed through 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 the conditions of gel filtration HPLC analysis II, and the estimated molecular weight and peak size were measured. The molecular weight was approximately 1 x 10 3 Fractions were collected at various intervals and lyophilized to obtain multiple neutral galactan fractions (fraction NG).

[0255] The catalytic activity, stereoselectivity, average molecular weight, and arabinose content of each fraction NG are shown in Figs. 5 to 6. The catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method III", the average molecular weight was measured according to "Gel Filtration HPLC Analysis Method II", and the arabinose content was measured according to "Monosaccharide Composition Analysis Method". Figs. 5(a) and (b) are graphs plotting the estimated molecular weight and catalytic activity of the neutral galactan fraction, respectively. When the estimated molecular weight was 3×10 3 or more, a tendency was observed that the higher the estimated molecular weight, the higher the catalytic activity. The catalytic activity reached a plateau around an estimated molecular weight of 6×10 3 . It was found that the stereoselectivity of the catalyst decreased significantly when the estimated molecular weight was 3000 or less. Also, the results of the monosaccharide composition analysis are shown in Fig. 6. As shown in Fig. 6, a tendency was observed that the higher the estimated molecular weight, the higher the arabinose content. In the fraction around 2.5×10 3 to 7×10 3 , the arabinose content was about 5%.

[0256] Example A10: Purification of Low Molecular Weight Polysaccharide Fraction (NG) Using HILIC-HPLC (1) Estimation of Degree of Polymerization in HILIC-HPLC Analysis An oligosaccharide (synthetic Gal9) with a degree of polymerization (DP) of 9 consisting of β1,4-linked galactose 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 about 0.4 mU / mg, which was comparable to commercially available D-galactose and showed almost no catalytic activity, and the stereoselectivity could not be confirmed either.

[0257] The fraction prepared in Example A9 (estimated molecular weight: 1 to 2×10 3 ) was analyzed under the following HILIC analysis conditions.

[0258] <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: 10 μL Detector: RID

[0259] The chromatogram showed a sawtooth shape in which peaks detected with high sensitivity (major peaks) and peaks detected with low sensitivity (minor peaks) eluted alternately multiple times. Since the peak of synthetic Gal9 overlapped with one of the major peaks, that peak was named "9mer", and based on this peak, the major peaks were numbered with integers in the order of elution, and the minor peaks sandwiched between the major peaks were numbered by adding ".5" to the number of the major peak that eluted immediately before. For example, the minor peak that eluted immediately before "9mer" (synthetic Gal9) is "8.5mer", and the major peak that eluted immediately before "8.5mer" is "8mer".

[0260] (2) HILIC-HPLC fractionation of "Fraction NG3 - 4 kDa" The fraction with a molecular weight of 3 - 4 × 10 3 obtained in Example A9 (Fraction NG3 - 4 kDa) was fractionated under the following HILIC fractionation conditions. The sample was dissolved in purified water to a concentration of 300 mg / mL, the supernatant was obtained by centrifugation, and filtered through a 0.2 μm diameter filter. The corresponding fractions were mixed, acetonitrile was removed by an evaporator, and lyophilization was performed. Under the HILIC-HPLC analysis conditions described in (1) above, the obtained fraction was analyzed under the condition that the eluent was changed to Acetonitrile / Purified water = 62 / 38, and the peaks were numbered in the same manner.

[0261] <HILIC-HPLC Fractionation 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: 10ml / min Column temperature: 30℃ Sample concentration: 1 mg / mL Injection volume: 100μL (=30mg) Fraction volume: 10 mL Detector: RID

[0262] 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) shows the chromatogram of the HILIC-HPLC preparative fraction "Fraction NG 3-4 kDa," and Figure 7(b) is a graph showing the chromatograms obtained by HILIC-HPLC analysis of each fraction after preparative separation. Each fraction showed a single peak in HPLC analysis. [Table 35]

[0263] (3) HILIC-HPLC separation of "Fraction NG 2-3 kDa" The molecular weight obtained in Example A9 is 2 to 3 × 10 3 This fraction (fraction NG 2-3 kDa) 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).

[0264] 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 same conditions as above. 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 same HILIC-HPLC analysis conditions as described in (1) above, except that the eluent was changed to acetonitrile / purified water (62 / 38), and each peak was numbered.

[0265] 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 performed 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) shows a graph overlaid with chromatograms of the HILIC-HPLC analysis of each fraction after separation. Each fraction showed a single peak in HPLC analysis.

[0266] [Table 36]

[0267] (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 [M+Na + ] or [M+K +It was considered to be the molecular ion peak of . Also, since the arabinose content in the isolated polysaccharide was 6% or less, it was estimated that it was composed only of galactose or contained 1 or 2 molecules of arabinose in one molecule. Therefore, based on the m / z value of the molecular ion peak, the number of galactose and arabinose molecules and the degree of polymerization (DP) in one molecule were estimated.

[0268] The estimated results of the degree of polymerization of each fraction are shown in Table 37. The sugar contained in the fraction of the major peak was mainly composed only of galactose, and the degree of polymerization (DP) and the peak number were in agreement. Also, the sugar contained in the fraction of the minor peak was mainly a polysaccharide containing 1 molecule of arabinose and the rest being galactose, and the degree of polymerization (DP) was in agreement with the value obtained by adding 0.5 to the peak number.

Table 37

[0269] (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 fractionation shown in Tables 35 to 37 are shown in Fig. 9.

[0270] The catalytic activity tended to be more excellent as the degree of polymerization was higher. The stereoselectivity tended to be more excellent as the degree of polymerization was higher between DP11 and DP15, and was comparable in the range of DP15 or more. From the viewpoint of stereoselectivity, it was considered that when the degree of polymerization was 13 or more, the practicality was higher.

[0271] (6) ODS-HPLC Fractionation 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 obtained filtrate was fractionated by ODS-HPLC under the following conditions. The corresponding fractions were mixed, MeOH was removed by an evaporator, and lyophilization was performed.

[0272] <ODS-HPLS Fractionation Conditions> Column: Open column equipped with a guard column (Mightysil RP-18GP Aqua, 5 μm, 10 mm I.D. × 250 mm) Eluent: Methanol / purified water (0.5 / 99.5) Injection volume: 100 μL (= 1 mg) Flow rate: 4 mL / min Column temperature: 40 °C Fractionation volume: 4 mL Detector: RID

[0273] The chromatogram of ODS-HPLC fractionation is shown in Figure 10. The peaks surrounded by the broken line were fractionated and named 19.5-1 to 8 in the order of elution. The fractionated fractions were each concentrated with an evaporator and freeze-dried to obtain samples "19.5-1 to 8". When each sample was analyzed under 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 superimposed.

[0274] <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: 10 μL Flow rate: 1.0 mL / min Column temperature: 40 °C Detector: RID

[0275] The catalytic activity, stereoselectivity, and molecular ion peak of each fraction obtained are shown in Table 38. The 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". Among the 19.5 mers, there was a polysaccharide (19.5-3) composed of 19 molecules of galactose and 1 molecule of arabinose, which showed a single peak in HPLC.

Table 38

[0276] Example A11: ODS-HPLC Fractionation The fraction BG2 (20 mg) obtained in Example A7 above was dissolved in 400 μL of purified water, and ODS-HPLC fractionation was performed under the following conditions.

[0277] <ODS-HPLC Fractionation Conditions> Column: YMC-Pack ODS-AQ (6 mm I.D. × 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.)

[0278] The fractionation chromatogram is shown in Fig. 12. As shown in Fig. 12, the fractions corresponding to peaks A to I were collected, and after repeating this a plurality of times, each was freeze-dried to obtain powders of 5 to 13 mg each. Fig. 13 shows the chromatograms when the respective fractions before and after fractionation were analyzed by ODS-HPLC. The analysis conditions are as follows. The fractions C to I corresponding to peaks C to I were all single peaks.

[0279] <Analysis Conditions> Column: YMC-Pack ODS-AQ (250 mm × 4.6 mm I.D. (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.)

[0280] For fractions D, E, and F, monosaccharide composition analysis was performed 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. From the analysis results, it was found that fractions D, E, and F are polysaccharides composed only of 20, 21, and 22 galactoses, respectively.

[0281]

Table 39

[0282] Similarly, the fraction BG3 obtained in Example A7 was fractionated by ODS-HPLC to obtain fractions corresponding to DP16 to DP19.

[0283] As described above, since DP16 to 25 were obtained respectively, the catalytic activity and stereoselectivity of each fraction were measured by "Catalytic Activity Measurement Method III". The results are shown in Fig. 14. Fig. 14(a) is a graph showing the catalytic activity of DP16 to 25, and Fig. 14(b) is a graph showing the stereoselectivity of DP16 to DP25. In the range of DP20 to DP25, the catalytic activity was high, and the higher the degree of polymerization, the higher the catalytic activity tended to be. Also, in the range of DP16 to DP20, the higher the degree of polymerization, the higher the stereoselectivity tended to be, and DP20 to DP25 showed the same degree of stereoselectivity.

[0284] Example A12: Modification of the reducing end of the low molecular weight polysaccharide fraction (1) Ethyl 4-aminobenzoate (ABEE) modification and tert-butyl 4-aminobenzoate (ABtB) modification of fractions BG1 to 5 Sodium cyanoborohydride (35 mg) and ABEE (165 mg) or ABtB (193 mg) were placed in a screw tube, methanol (350 μL) and acetic acid (41 μL) were added, and the mixture was stirred by vortexing and heated at about 60 °C to dissolve to prepare the derivatization reagent.

[0285] Each solution (10 mg / mL, 10 μL) of BG1 to 5 obtained in Example A6 was placed in a 1.5 mL microtube, the derivatization reagent (40 μL) was added, and the mixture was heated at 80 °C for 1 hour. Purified water (500 μL) was added, and the mixture was extracted with chloroform, and the aqueous layer was analyzed by HPLC under the following conditions.

[0286] <HPLC analysis> Column: TSKGel ODS120H (manufactured by Tosoh Corporation) Eluent: sodium borate buffer (pH 9.0) / acetonitrile (91 / 9) Flow rate: 1.0mL / min Column temperature: 35℃ Detector: UV (detection wavelength: 310 nm)

[0287] As a result of HPLC analysis, ABEE-modified BG1-3 each showed a broad single peak, while ABEE-modified BG4-5 each showed multiple peaks. 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.

[0288] (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).

[0289] NG-ABEE and the unmodified sample (NG) were analyzed by the "Gel Filtration HPLC Analysis II" method. The results are shown in Figure 15. Detection was performed by both RID and UV (detection wavelength: 310 nm). NG-ABEE was confirmed to have absorbance at a wavelength of 310 nm by UV detection, and RID detection revealed that the retention times of NG and NG-ABEE were almost the same.

[0290] 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. [Table 40]

[0291] (3) ABEE synthesis and catalytic activity of low molecular weight polysaccharide fractions In a similar manner, the fraction AM-LLMW obtained in Example A7 was converted to ABEE.

[0292] When the aqueous layer was analyzed by the "Gel Filtration HPLC Analysis II" method, a peak with absorption at a wavelength of 310 nm was detected. It was confirmed that fraction AM-LLMW could also be converted to ABEE.

[0293] Example B1: NMR Analysis Under the following conditions, AL-LMW 1 H-NMR and 13 C-NMR was measured. Solvent: Deuterium oxide (DO) NMR measurement device: Bruker AVANCE II 400 (manufactured by Bruker) frequency: 1 400MHz for H-NMR, 13 100MHz for C-NMR

[0294] 1 The H-NMR spectrum is shown in Figure 16. 13 The 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)). The NMR spectrum of AL-LMW was similar to the value 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 linked via a β1,4 bond. Furthermore, no peaks corresponding to olefins or carbonyl groups were observed in the NMR spectrum.

[0295] Example B2: Multi-step derivatization of fraction NG3-4 kDa and analysis of glycan binding positions The 11-mer, 11.5-mer, 15-mer, 15.5-mer, 19.5-mer, or 20-mer prepared from the NG3-4 kDa fraction obtained in Example A10 was used as a sample, and multi-step derivatization (methylation, monosaccharide conversion, alditol conversion, and acetylation) was performed, and the binding position of the glycan was analyzed from the difference in molecular weight. (Step 1) Methylation A sample (1 mg) was weighed into a 2 mL screw-cap tube, suspended in dimethyl sulfoxide (500 μL) and sodium hydroxide (50 mg) ground into powder in a mortar, and stirred 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) Monosaccharide synthesis The methylated composition was added with 100 mM sodium thiosulfate aqueous solution (300 μL) and dichloromethane (300 μL), and the organic layer was collected. The organic layer was washed with 100 mM sodium thiosulfate aqueous 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) Alditolation The composition after monosaccharification was cooled to room temperature and then freeze-dried, and sodium borodeuteride (20 mg) and 2M aqueous ammonia (500 μL) were added and dissolved, followed by shaking 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.

[0296] <GC-MS Conditions> Measuring instrument: GC2010 / GCMS-QP2010 (manufactured by Shimadzu Corporation) Analysis column: DB-225MS (20 m × 0.25 mm I.D., 0.25 μm) Carrier gas: Helium Column temperature: Maintained at 140°C for 1 minute, then heated to 220°C at a rate of 20°C / min and maintained at 220°C for 25 minutes.

[0297] Figure 18 is a GC spectrum obtained by detecting m / z 118 after derivatizing 19.5mer and 20mer. Peaks detected at m / z 118 are generally peaks frequently appearing in the mass spectrum, indicating that some hydrogen atoms of partially methylated alditol acetate derived from hexose have been replaced by deuterium (see J. Biol. Chem., 293(30), 11955 - 11965 (2018)). Also, according to the spectrum library described later, many peaks derived from arabinose are detected at m / z 118.

[0298] In the GC-MS analysis, three peaks with retention times of 9.450 minutes (a), 9.583 minutes (b), and 12.517 minutes (c) were observed by detecting m / z 118. When the mass spectra of each peak were compared with the data of the spectrum library (https: / / glygen.ccrc.uga.edu / ccrc / specdb / ms / pmaa / pframe.html) provided by the Complex Carbohydrate Research Center of the University of Georgia, it was found that they corresponded to (a) 1,4-arabinopyranose or 1,5-arabinofuranose, (b) terminal galactopyranose, and (c) 1,4-galactopyranose in order of increasing retention time. From these results, it was estimated that 19.5mer and 20mer (neutral galactan) are sugar chains in which galactose is linearly linked, have no branched chains, and arabinose is inserted between sugar chains mainly composed of 1,4-linked galactose.

[0299] Example C: Examination 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 a "FASTKIT Slim Soy" (manufactured by Nippon Meat Packers Co., Ltd.) according to the manufacturer's instructions. Purified water was added to the okara to extract the soybeans, which were then centrifuged, and the resulting supernatant was subjected to analysis.

[0300] 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 also measured at a five-fold increased sample concentration. The results are shown in Table 41. The polysaccharide fractions had lower soy allergen levels than the water-soluble soy polysaccharides, and the low molecular weight polysaccharide fractions were found to have undetectable levels. [Table 41] ++: Positive bands appear dark. +: Positive bands are faint. -: No positive bands observed.

[0301] Example C2: Resistance to gelation of allergens Twenty milligrams 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 100 μL. After leaving the container at room temperature for one day, the container was inverted. If the solution did not flow, it was considered 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.

[0302] Example C3: Stereoselectivity Comparison Stereoselectivity was evaluated (n = 5) using polysaccharide samples (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 significant.

[0303] 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. [Table 42]

[0304] Example C4: Improved product recovery with 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 vessel, and 300 μL of a toluene solution of the substrate (containing 10 v / v% epoxycyclohexane and 10.8 v / v% cyclopropylamine) was added. The mixture was stirred 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 performed.

[0305] The content of the product (2-cyclopropylaminocyclohexanol) in the recovered toluene layer was measured by GC analysis according to "Method I for Measuring Catalytic Activity."

[0306] The results are shown in Table 43. Neither the water-soluble soy polysaccharide nor AM-LLMW showed any change in the conversion rate, the recovery amount of the product, or the optical purity even after repeated use. On the other hand, when the water-soluble soy polysaccharide was repeatedly used, part of the reaction solution gelled and adhered to the inner wall of the container, making it difficult to recover the toluene layer and resulting in a small recovery amount of the product. Fig. 19 is a photograph showing the appearance of each reaction vessel before recovering the toluene layer. The left shows the reaction vessel using the water-soluble soy polysaccharide, and the right shows the reaction vessel using AM-LLMW. In Fig. 19, the solid-line arrow indicates the position of the liquid surface of the toluene layer, and the dashed-line arrow indicates the position of the interface between the aqueous layer and the toluene layer.

Table 43

[0307] Example C5: High-temperature reactivity Using soy five S-DN or LP-LMW, the reaction temperature was changed to 40 °C, 50 °C, 60 °C, and 70 °C in Activity Measurement Method V, and the catalytic activity and stereoselectivity were measured. The results are shown in Table 44. Both LP-LMW and the water-soluble soy polysaccharide reached their maximum at 60 °C. The stereoselectivity was hardly affected by the temperature. At any temperature, LP-LMW maintained a specific activity 1.5 to 2 times higher than that of soy five S-DN.

Table 44

[0308] Example D: Production of amino alcohol The racemic amino alcohol, which serves as a reference sample for the synthesized product, was analyzed by GC and HPLC under the following conditions. The analysis conditions are shown in Tables 45 and 46.

[0309] <GC analysis conditions>

Table 45

[0310] <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 [Table 46]

[0311] 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. The mixture was stirred at 50°C for 48 hours. The reaction mixture 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 = 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 min [epoxide: 4.1 min, amine: 13.3 min] Analysis conditions α 42.1 minutes: (1S,2S) isomer, 45.6 minutes: (1R,2R) isomer

[0312] 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. 86 mg of 3,4-epoxytetrahydrofuran and 8.5 mg of lithium chloride were added sequentially, and the mixture was vigorously stirred in a 50°C oil bath 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 min [epoxide: 6.2 min, amine: 8.8 min] Analysis conditions χ 38.2 minutes: (3S,4R) isomer, 41.1 minutes: (3R,4S) isomer [Amine: 21.2 minutes]

[0313] Reference Example 3: Synthesis of trans-2,2-dimethyl-6-(benzylamino)-1,3-dioxepan-5-ol A reaction was carried out in the same manner as in Reference Example 2 using 144 mg of 4,4-dimethyl-3,5,8-trioxabicyclo[5.1.0]octane and 107 mg of benzylamine, and the reaction mixture was purified by silica gel column chromatography (ethyl acetate:hexane:triethylamine=50:1:1, gel 30 mL) to obtain 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]

[0314] 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 min [epoxide: 10.1 min, amine: 13.3 min] Analysis conditions ε 25.4 minutes: (5S,6R) isomer, 27.4 minutes: (5R,6S) isomer

[0315] Reference Example 5: Synthesis of trans-2,2-dimethyl-6-(2-propynylamino)-1,3-dioxepan-5-ol The procedure of Reference Example 2 was repeated, except that 144 mg of 4,4-dimethyl-3,5,8-trioxabicyclo[5.1.0]octane and 112 mg of propargylamine were used and purified 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 min, 32.4 min [epoxide: 5.5 min]

[0316] Reference Example 6: Synthesis of trans-2,2-dimethyl-6-(cyclopropylamino)-1,3-dioxepan-5-ol The procedure of Reference Example 2 was repeated, 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 purified 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 min, 32.4 min [epoxide: 5.5 min]

[0317] 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 50.6 mg of (1R,2R)-2-aminocyclohexanol hydrochloride, 38 μL of acetic acid, and 78.6 mg of sodium cyanoborohydride were added sequentially under vigorous stirring. The mixture was then stirred for 19 hours. The reaction mixture was concentrated and extracted three times with chloroform. The combined organic layer was washed 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 condition α 22.2 minutes (>99% ee)

[0318] 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 mixture 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 condition β 45.7 minutes (>99% ee)

[0319] 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-aminotetrahydrofuran-3-ol, 19.2 μL of acetic acid, and 45.5 mg of sodium cyanoborohydride were added sequentially and stirred vigorously for 69 hours. The reaction mixture was concentrated and extracted three times with chloroform. The organic phase was washed sequentially 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 (10.0% yield). Analysis condition β 27.6 minutes (>99% ee)

[0320] Reference Example 10: Synthesis of (3S,4R)-4-((4-methoxybenzyl)amino)tetrahydrofuran-3-ol A reaction was carried out in the same manner as in Reference Example 9 using 27.2 mg of 4-methoxybenzaldehyde and 20.6 mg of (3S,4R)-4-amino-tetrahydrofuran-3-ol, and the mixture was purified by PTLC (CHCl3:MeOH:Et3N = 20:1:1) to obtain 5.9 mg of the title compound as a yellow powder (yield 16.5%). Analysis conditions: χ 36.6 min (>99% ee)

[0321] 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. 20 μL of 0.5 M titanium tetraisopropoxide in anhydrous toluene was added under vigorously stirring. After 10 minutes of stirring, 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 stirred vigorously for 18.5 hours. 2 mL of ethyl acetate was added to the reaction mixture, which was then vacuum 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, 30 mL of gel) to obtain 128.8 mg of the title compound as a white powder (51.3% yield). Analysis conditions δ 28.3 min (95.3% ee)

[0322] 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, the reaction and extraction were carried out in the same manner as in Reference Example 11, and the 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)

[0323] <Production of 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 through 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 polysaccharide.

[0324] <Catalytic activity of water-soluble soybean polysaccharides and LMW toward various substrates> The results are shown in Tables 47 to 49. [Table 47] Bn: benzyl group, PMB: p-methoxybenzyl group, Not tested: not tested [Table 48] Bn: benzyl group, PMB: p-methoxybenzyl group, Not tested: not tested [Table 49] Bn: benzyl group, PMB: p-methoxybenzyl group, Not tested: not tested

[0325] <Reaction of various amino alcohols produced by 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 saturated aqueous sodium chloride or saturated aqueous potassium chloride was added. The reaction was carried out at 37 ± 1 °C with vigorous stirring for 3 days. The reaction solution was collected 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 resulting amino alcohol.

[0326] 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. This was analyzed to confirm the retention times of the enantiomers of the product. [Table 50]

[0327] <Production of various amino alcohols using polysaccharide fractions 3> 10 mg of AL-LMW was weighed into a 2 mL glass vial, 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. [Table 51]

[0328] Example D2: Aminoalcohol formation reaction 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 mixture 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 mixture were calculated and are listed 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, the mixture was stereoselectively converted to 2-cyclopropylaminocyclohexanol. [Table 52]

[0329] Example E: Catalytically inactive galactans The catalytic activity of commercially available polysaccharides containing arabinogalactans was measured according to "Catalytic Activity Measurement Method I" or "Catalytic Activity Measurement Method III."

[0330] 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. [Table 53]

[0331] Example F1: Preparation of Gal Donor The 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-Tolu Oil 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 [ka]

[0332] [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. The mixture was stirred at room temperature for 1 day. Triethylamine (7.4 mL) was added to the reaction mixture to neutralize it. The precipitated solid was filtered off, and 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, CDCl3): 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 (2 d, J = 11.9 Hz, CH2Ph), 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).

[0333] [2] Synthesis of 4-Methoxyphenyl 3-O-benzyl-4,6-O-benzylidene-2-Op-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, then cooled to room temperature. Methanol (100 mL) was added to decompose the excess reagent, and the reaction mixture was concentrated under reduced pressure. The residue was dissolved in methylene chloride and washed with 2 M hydrochloric acid and 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 recrystallized from methylene chloride-isopropyl ether to give compound S3 (286 g, yield 96%). 1 H-NMR (400MHz, CDCl3): 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, CH2Ph), 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).

[0334] [3] Synthesis of 4-Methoxyphenyl 3,6-di-O-benzyl-2-Op-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 mixture was diluted with methylene chloride and washed with water, saturated sodium bicarbonate water, and saturated brine, successively. 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 recrystallized from methylene chloride-isopropyl ether to give compound S4 (139 g, 93% yield). 1 H-NMR (400MHz, CDCl3): 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, CH2Ph), 4.60 (s, 2 H, CH2Ph), 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).

[0335] [4] Synthesis of 5-tert-butyl-2-methylphenyl 3,6-di-O-benzyl-1-thio-2-Op-toluoyl-β-D-galactopyranoside (Compound S5) Compound S4 (60 g, 103 mmol) was dissolved in methylene chloride (900 mL), 5-tert-butyl-2-methylbenzenethiol (93 mL, 5 equivalents) and boron trifluoride diethyl ether complex (6.4 mL, 0.5 equivalents) were added, and the mixture was stirred at room temperature for 15 minutes. The reaction mixture was then 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, 70% yield). 1 H-NMR (400MHz, CDCl3): 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, CH2Ph), 4.58 (s, 2 H, CH2Ph), 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).

[0336] [5] Synthesis of 5-tert-butyl-2-methylphenyl 3,6-di-O-benzyl-4-O-chloroacetyl-1-thio-2-Op-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 mixture 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, CDCl3): 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, COCH2Cl), 4.44, 4.58, 4.66 (3 d, 4 H, J = 12.4 Hz, 2 CH2Ph), 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, 2H, ortho- of COPhMe).

[0337] Example F2: Preparation of Gal(2mer) derivatives [ka]

[0338] [1] Synthesis of 4-methoxyphenyl (3,6-di-O-benzyl-4-O-chloroacetyl-2-Op-toluoyl-β-D-galactopyranosyl)-(1-4)-3,6-di-O-benzyl-2-Op-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. 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 subjected to silica gel column chromatography (elution solvent: toluene:ethyl acetate = 16:1) to obtain compound S7 (49.8 g, 65% yield). 1H-NMR (400MHz, CDCl3): 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, COCH2Cl), 4.28 (d, 1 H, J = 2.7 Hz, H-4a), 4.37-4.67 (7 d, 8 H, 4 CH2Ph), 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).

[0339] [2] Synthesis of 5-tert-butyl-2-methylphenyl (3,6-di-O-benzyl-4-O-chloroacetyl-2-Op-toluoyl-β-D-galactopyranosyl)-(1-4)-3,6-di-O-benzyl-1-thio-2-Op-toluoyl-β-D-galactopyranoside (Compound S8) Compound S7 (25 g, 22.3 mmol) was dissolved in methylene chloride (500 mL), 5-tert-butyl-2-methylbenzenethiol (20 mL, 5 equivalents) and boron trifluoride diethyl ether complex (1.4 mL, 0.5 equivalents) were added, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was then 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: toluene:ethyl acetate = 20:1) to give compound S8 (13.6 g, 52% yield). 1 H-NMR (400MHz, CDCl3): 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, COCH2Cl), 4.31 (d, 1 H, J = 2.3 Hz, H-4a), 4.35-4.67 (m, 8 H, 4 CH2Ph), 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).

[0340] [3] Synthesis of 4-Methoxyphenyl (3,6-di-O-benzyl-2-Op-toluoyl-β-D-galactopyranosyl)-(1-4)-3,6-di-O-benzyl-2-Op-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 mixture 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, 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, CDCl3): 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 CH2Ph), 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).

[0341] Example F3: Preparation of Gal(4mer) derivative [ka]

[0342] [1] Synthesis of 4-methoxyphenyl (3,6-di-O-benzyl-4-O-chloroacetyl-2-Op-toluoyl-β-D-galactopyranosyl)-(1-4)-(3,6-di-O-benzyl-2-Op-toluoyl-β-D-galactopyranosyl)-(1-4)-(3,6-di-O-benzyl-2-Op-toluoyl-β-D-galactopyranosyl)-(1-4)-3,6-di-O-benzyl-2-Op-toluoyl-β-D-galactopyranoside (Compound S10) Compound S8 (16.4 g, 12.5 mmol) and compound S9 (18.9 g, 1.4 equiv.) 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 equiv.) and dimethyl(methylthio)sulfonium triflate (50 wt% molecular sieves mixture, 12.9 g, 2 equiv.) 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 sodium bicarbonate water. 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 subjected to silica gel column chromatography (elution solvent: toluene:ethyl acetate = 15:1) to obtain compound S10 (20.0 g, 78% yield). 1 H-NMR (400MHz, CDCl3): 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, COCH2Cl), 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).

[0343] [2] Synthesis of benzyl (3,6-di-O-benzyl-4-O-chloroacetyl-2-Op-toluoyl-β-D-galactopyranosyl)-(1-4)-(3,6-di-O-benzyl-2-Op-toluoyl-β-D-galactopyranosyl)-(1-4)-(3,6-di-O-benzyl-2-Op-toluoyl-β-D-galactopyranosyl)-(1-4)-3,6-di-O-benzyl-2-Op-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 stirred at 0 °C for 3 hours. The reaction mixture was extracted with ethyl acetate and washed with water, saturated sodium bicarbonate solution, and saturated brine, successively. The organic layer was dried over magnesium sulfate, the desiccant was removed by filtration, 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 crude compound S11 (10 g). This crude product 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 stirred at 0 °C for 3 hours. The reaction mixture was subjected to silica gel column chromatography (eluent: toluene: ethyl acetate = 15:1) to obtain crude 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 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, the desiccant was filtered off, and the solvent was evaporated under reduced pressure. The resulting residue was subjected to silica gel column chromatography (eluent: hexane: ethyl acetate = 2:1) to obtain compound S13 (8.2 g, 75% yield). 1H-NMR (400MHz, CDCl3): 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, COCH2Cl), 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).

[0344] Example F4: Glycan chain extension [1] Preparation of Gal oligomer receptor [ka]

[0345] Gal oligomer derivatives (compounds S13, S15, S17, S19, S21, S23, S25, S27, and S29) were dissolved in ethyl acetate (7.5 mL / g of receptor) and ethanol (7.5 mL / g of receptor), and 1,4-diazabicyclo[2.2.2]octane (10 equivalents) was added. The mixture was stirred at 50 °C for 2 h. The reaction mixture was cooled to room temperature, diluted with ethyl acetate, and washed with 2 M hydrochloric acid and saturated aqueous sodium bicarbonate. The organic layer was dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The resulting residue was purified by silica gel column chromatography (elution solvent: toluene:ethyl acetate = 9:1) to obtain Gal oligomer receptors (compounds S14, S16, S18, S20, S22, S24, S26, S28, and S30). Compound S14: Yield: 8.0 g (89%). 1H-NMR (400MHz, CDCl3): 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: The yield was 7.7g (the yield of compound S14 was 59%). 1 H-NMR (400MHz, 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: The yield is 6.6g (the yield of compound S16 is 65%). 1H-NMR (400MHz, CDCl3): 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: The yield is 4.9g (the yield of compound S18 is 60%). 1 H-NMR (400MHz, CDCl3): 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 MALDI-TOF-MS: Calcd for C 287 H 288 O 61 Na m / z [M+Na] + : 4732.9, Found: 4732.5. Compound S22: The yield was 3.1g (the yield of compound S20 was 52%). 1H-NMR (400MHz, CDCl3): 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: The yield is 1.8g (the yield of compound S22 is 52%). 1 H-NMR (400MHz, CDCl3): 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: The yield is 800 mg (the yield of compound S24 is 40%). 1H-NMR (400MHz, CDCl3): 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 MALDI-TOF-MS: Calcd for C 455 H 456 O 97 Na m / z [M+Na] + : 7499.5, Found: 7498.0. Compound S28: The yield was 360 mg (the yield of compound S26 was 40%). 1 H-NMR (400MHz, CDCl3): 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 MALDI-TOF-MS: Calcd for C 511 H 512 O 109 Na m / z [M+Na] + : 8420.6, Found: 8421.0. Compound S30: The yield was 210 mg (the yield of compound S28 was 53%). 1H-NMR (400MHz, CDCl3): 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 MALDI-TOF-MS: Calcd for C 567 H 568 O 121 Na m / z [M+Na] + : 9341.7, Found: 9342.0.

[0346] [2] Glycosylation of Gal oligomer receptors [ka] 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) were 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 mixture was filtered through Celite, and the filtrate was washed with saturated sodium bicarbonate water. The organic layer was dried over magnesium sulfate, the desiccant was removed by filtration, and 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: 11H-NMR (400 MHz, CDCl3): 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, COCH2Cl), 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 1H-NMR (400 MHz, CDCl3): 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, COCH2Cl), 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 11H-NMR (400 MHz, CDCl3): 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, COCH2Cl), 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 COPhMe). MALDI-TOF-MS: Calcd for C 289 H 289 ClO 62 Na m / z [M+Na] + : 4808.9, Found: 4808.8. Compound S21: 1 1H-NMR (400 MHz, CDCl3): 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, COCH2Cl), 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 COPhMe). MALDI-TOF-MS: Calcd for C 345 H 345 ClO 74 Na m / z [M+Na] + : 5729.3, Found: 5728.9. Compound S23: 1H-NMR (400 MHz, CDCl3): 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, COCH2Cl), 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 COPhMe). 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 (400 MHz, CDCl3): 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, COCH2Cl), 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 COPhMe). MALDI-TOF-MS: Calcd for C 457 H 457 ClO 98 Na m / z [M+Na] + : 7570.0, Found: 7570.5. Compound S27: 11H-NMR (400 MHz, CDCl3): 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). MALDI-TOF-MS: Calcd for C 513 H 513 ClO 110 Na m / z [M+Na] + : 8497.1, Found: 8498.7. Compound S29: 1 1H-NMR (400 MHz, CDCl3): 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, H-2), 5.65 (d, 1 H, J = 3.2 Hz, H-4), 6.91 - 7.38 (m, aromatic), 7.71 - 7.97 (m, 40 H, 20 ortho- of COPhMe). MALDI-TOF-MS: Calcd for C 569 H 569 ClO 122 Na m / z [M+Na] + : 9418.1, Found: 9418.2.

[0347] Example F5: Deprotection

Chem.

[0348] The NMR analysis data was similar to the value derived from (→4)-β-D-Galp-(1→) reported in the literature (Food Chemistry, 242, 211-216(2018)), supporting the idea that compound S31 is composed of 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.

[0349] 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, 200 μL of 25 mM sodium chloride aqueous solution was added, and the mixture was freeze-dried. Catalytic activity and stereoselectivity were measured according to "Catalytic Activity Measurement Method III." The results are shown in Table 54. A comparison of compound S31 with BG3 fraction, which contains polysaccharides prepared from water-soluble soybean polysaccharides in the same manner as in Example A7(6) and has an average molecular weight similar to that of compound S31, revealed that the catalytic activity and stereoselectivity were comparable or superior to those of compound S31. [Table 54]

Claims

1. 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.

2. 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.

3. A composition comprising the polysaccharide of claim 1 or 2.

4. 4. The composition of claim 3 for use as a catalyst.

5. 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.

6. A compound represented by formula (1) in the presence of the polysaccharide according to claim 1 or 2, 【Chemical 1】 wherein X is —O—, —NR—, or —S—; R is a hydrogen atom, an optionally substituted C １－６ alkyl group, optionally substituted C ３－６ cycloalkyl group, optionally substituted C ２－６ Alkenyl group, optionally substituted C ３－６ Cycloalkenyl group, optionally substituted C ２－６ Alkynyl group, optionally substituted C ６－１０ aryl group, optionally substituted C １－６ alkylcarbonyl group, optionally substituted C ６－１０ arylcarbonyl group, optionally substituted C １－６ Alkylsulfonyl group or C ６－１０ is an 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, optionally substituted C ３－６ cycloalkyl group, optionally substituted C ２－６ Alkenyl group, optionally substituted C ３－６ Cycloalkenyl group, optionally substituted C ２－６ Alkynyl group or optionally substituted C ６－１０ is an aryl group, R ２ and R ３ may be bonded to each other to form a cyclic structure represented by (1a). 【Chemistry 2】 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, optionally substituted C ３－６ cycloalkyl group, optionally substituted C ２－６ Alkenyl group, optionally substituted C ３－６ Cycloalkenyl group, optionally substituted C ２－６ Alkynyl group or optionally substituted C ６－１０ is an aryl group, R ２ａ and R ３ａ are each independently C １－６ Alkylene group, C １－６ Oxyalkylene group, C ３－６ Cycloalkylene group, C ２－６ Alkenylene group, C ３－６ Cycloalkenylene group, C ２－６ Alkynylene group or C ６－１０ It is an arylene group.] reacting a compound represented by formula (2) with 【Chemistry 3】 [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, optionally substituted C ３－６ cycloalkyl group, optionally substituted C ２－６ Alkenyl group, optionally substituted C ３－６ Cycloalkenyl group, optionally substituted C ２－６ Alkynyl group or optionally substituted C ６－１０ is an aryl group, R ５ and R ６ 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.] 【Chemistry 4】 [In the formula, R ５ａ and R ６ａ each represents an optionally substituted C １－６ Alkylene group, optionally substituted C １－６ Oxyalkylene group, optionally substituted C ３－６ Cycloalkylene group, optionally substituted C ２－６ Alkenylene group, optionally substituted C ３－６ Cycloalkenylene group, optionally substituted C ２－６ Alkynylene group or C ６－１０ It is an arylene group.] A method for producing a compound represented by formula (3), comprising the step of obtaining a compound represented by formula (3). 【Chemistry 5】 [In the formula, X, Y, R １ , R ２ , R ３ , R ４ and R ５ is as defined in formula (1) or (2).

7. (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:

8. 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 differences in solubility, treatment with an anion exchange resin, gel filtration chromatography, HPLC, or any combination thereof.

9. 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). 【Chemistry 6】 [In the formula, R Ａ is an optionally substituted alkylcarbonyl group, and R Ｂ is an optionally substituted aralkyl group, and R Ｃ is an optionally substituted arylcarbonyl group, and R Ｄ is an optionally substituted aryl group, and R Ｅ represents an optionally substituted aralkyl group, and n is an integer of 13 to 21.

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