TWI324607B - - Google Patents

Description

0) 0) 1324607 Rose, invention description (invention description should be stated: the technical field, prior art, content, embodiment and schematic description of the invention) TECHNICAL FIELD The present invention relates to a sugar chain aspartate derivative A method for producing a substance and a sugar chain aspartame derivative. Background Art In the past, a technique in which a sugar hydrolyzate is decomposed into a derivative by a sugar hydrolyzing enzyme is used for analysis of a structure of a sugar chain, and is analyzed on a milligram scale. However, since the individual sugar chain derivatives cannot be obtained in a large amount, the progress in the gram scale research technique is rather slow. Therefore, it is quite difficult to apply a derivative of a sugar chain to a synthetic study of a manufacturable drug. On the other hand, it is known that a glycopeptide can be obtained in a large amount from egg yolk (Biochimica et Biophysica Acta 1335 (1997) p 23 to 32). However, regarding the compound 1 shown in Fig. 1 and the sialic acid and galactose having a non-reducing end on the side of the branched sugar chain in the compound 1, there is no successful example showing that a large amount of warp can be obtained. An oxycarbonyl (Fmoc)-derived sugar chain derivative. Further, there have been cases in which a few sugar chains are separated from a small amount of protein in human blood, but if the sugar chain is used in the manufacture of a drug, the drug will have an HIV virus and a hepatitis virus which are harmful to the human body. The danger is equal, so there is still a technical problem in applying this sugar chain to medicines. Further, in the branched type sugar chain, a sugar chain having the same structure of the branched portion is produced, and a considerable number of manufacturing examples exist. There are three methods in its past technology. The first method consists of a naturally occurring glycoprotein 1324607 (2) Description of the invention 分离 Separation and purification of the aspartate-linked complex sugar chain. Representative examples thereof are, for example, T. Tamura, et al. Anal. Biochem., 1 994, 2 1 6, p 3 3 5-344 > VH Thomas, et al. Carbohydr. Res., 1998, 306, p 3 8 7-400 > KG Rice, et al., Biochemistry, 1 993, 32, p 7264-7270, etc. The advantage of these methods is that there is no need to synthesize sugar chains. However, it also has several shortcomings. For example, the aforementioned glycoprotein-derived sugar chain may result in a mixture of several randomly occurring sugar residues in the non-reducing terminal moiety, the sugar chain contained in the mixture, due to its physical and chemical properties. Very similar, so it is very difficult to separate individual sugar chains, and it is virtually impossible to obtain a single sugar chain in large quantities. Also, in order to obtain a larger amount (sugar chain), there are also examples of proteins in human blood (separation by fibrinogen: CHHokke. et al, Carbohydr. Res., 305 (1997), p 463-468, isolation of human serum iron transfer protein: M. Mizuno et al., J. Am. Chem. Soc., 1999, 121, p 284-290) to separate sugar chains. As mentioned earlier, proteins in human blood may be contaminated with HIV or hepatitis viruses, so this step must be done with great care. Therefore, it is difficult to use the obtained sugar chain and its derivatives for the development of pharmaceuticals. Further, even if the sugar chain can be obtained in a large amount, it is not limited to the structure, and there are no examples of substantially obtaining a sugar chain having a variety of structures and derivatives thereof. At K · G. Ri c e, et al., B i 〇 c hemi s tr y, 1 993, 32, p 7264-7270 , or Rice, et al., Neogly coconjugate,

Academic Press, 1 994, ISBN 0- 12-4405 85- 1 p 286-32 1 , using sugar hydrolyzing enzymes to remove sugar residues from the non-reducing ends of sugar chains, but 1324607 (3) [SeSem] A single structured sugar is obtained as a raw material, and therefore only stays at the level of analysis scale. E. Meinjohanns (J. Chem. Soc. Perkin Transl, 1998, p 549-560) et al., after obtaining compound 56 shown in Figure 5 from bovine fetuin (a glycoprotein derived from cattle), via Figure 3 The compound 10 shown in Figure 1 was synthesized as the compound 3 3 shown. In order to obtain the compound 56 of the initial starting material, the hydrazine decomposition reaction is utilized. This hydrazine has high toxicity, and when the derivative of the obtained sugar chain is used in a pharmaceutical product, there is a possibility that a trace amount of hydrazine may be mixed to cause a safety problem. Further, derivatives of the sugar chain of the compounds 5-6, 3 3 and 10 which are not bonded with sialic acid can be obtained only in a small amount. The second method is a method of chemically synthesizing sugar chains. Now, when the monosaccharide is combined by chemical synthesis, as exemplified by J. Seifert et al. Angew Chem Int. Ed. 2000, 39, p 531-534, it can be constructed to about 10 sugar units. The advantage of this method is that all sugar chain derivatives are theoretically available. However, due to the complexity of the steps, there is a disadvantage that it cannot be synthesized in large quantities. Further, even if it is necessary to synthesize a sugar chain having 10 or so sugar residues to a few milligrams, it takes almost a year. So far, although there are some examples of chemically synthesizing several sugar chains, many of them only stay at the stage of being able to synthesize several milligrams of the target sugar chain. The third method is a method of synthesizing a saccharide by combining an enzyme reaction and a chemical reaction. Representative examples thereof are reported by Carlo Unverzagt, Angew Chem Int. Ed. 1996, 35, p 2350-2353. This method is a method in which a sugar chain is constructed by chemical synthesis and then a sugar residue is attached to the sugar chain by an enzyme reaction to lengthen the sugar chain. However, in the long sugar chain lock 1324607

(4) The enzymes used in ISSiSSS are limited in their ability to be introduced into the sugar chain due to matrix specificity. Moreover, the number of chemical synthesis steps is numerous, which also causes a large amount of synthesis difficulties, so that only a small amount of final product can be obtained. Further, C. H. Lin (Bioorganic & Medicinal Chemistry, 1 995, p 1625-1630) et al. use the method reported by M_Koketsu et al.

Carbohydrate Chemistry, 1 9 9 5, 1 4 (6), ρ 83 3-84 1 ), the sialyl oligosaccharide peptide is obtained from the egg yolk, and then the sugar hydrolyzate and the sugar transfer enzyme are used, and the sugar chain is changed. The structure of the non-reducing end portion is shown in the figure. The sugar chain described has only one aspartate (Asn) residue on the non-reducing end portion. However, according to the method reported by J. Carbohydrate Chemistry, 1 995, 14 (6), ρ 83 3-84 1 , a mixture is still obtained, and the non-reducing end of the sugar chain has a bond other than aspartame. There are several amino acids such as lysine (on average, 2.5 amino acids are bonded). Therefore, it is not possible to obtain a single compound type saccharide derivative, and it is not taught that a large amount of each derivative can be obtained, and among the compounds of the respective derivatives, the sugar of the branched sugar chain of the branched sugar chain Residue _ is a case of random deletion. In the paper by C. H. Lin et al., there is no evidence that a single-product glycopeptide is obtained. Y. Ichikawa is described in Gly copeptide and Related Compounds (Marcel Dekker, Inc., 1997, ISBN 0-8247-953, 8, p. 79-205), starting with a tribranched complex sugar chain from the end, When the sugar hydrolyzing enzyme is sequentially treated, the sugar chain can be sequentially removed from the non-reducing end of the sugar chain to obtain various sugar bond derivatives. However, it does not mention how to separate individual sugar chains, and the synthesis is only uniform. Therefore, -10- 1324607 (5), even according to this method, a compound in which a sugar residue of a branched sugar chain of a branched sugar chain is randomly deleted is generally not available in a large amount for individual derivatives. Invention

According to the present invention, various isolated sugar chain aspartate derivatives which are very useful in the field of pharmaceutical development can be obtained in a large amount in comparison with the conventional methods. Further, an object of the present invention is to provide a method for producing a sugar chain aspartame and a method for producing a sugar chain, and to obtain a sugar chain aspartate by a step of producing a sugar chain aspartate derivative At the same time as the derivatives, various useful isolated sugar chain aspartames and sugar chains are obtained very easily and in large quantities compared to the conventional methods. Further, it is an object of the present invention to provide a novel sugar chain aspartame derivative, a sugar chain aspartate and a sugar chain. That is, the present invention relates to (1) a method for producing a sugar chain aspartame derivative derived from a sugar chain aspartame, which comprises:

(a) a step of obtaining a mixture of sugar chain aspartate derivatives, which is introduced into the sugar chain aspartate containing a mixture of one or more sugar chain aspartame, and is introduced into fat-soluble protection And (b) a step of isolating each of the sugar chain aspartate derivatives, which is a sugar chain aspartate contained in the sugar chain aspartate derivative mixture or the sugar chain aspartate derivative mixture a guanamine derivative, which is hydrolyzed to obtain a mixture, which is then subjected to color layer analysis; (2) A manufacturer of the sugar chain aspartate derivative as described in the above (1) - 11 - 1324607

(6) i invention description sheet method, which further comprises: (b) a step of using a glycolytic enzyme hydrolysis step (b) to separate the sugar chain aspartate derivative; (3) as described above (1) Or a method for producing a sugar chain aspartame derivative according to the above aspect, wherein the mixture comprising one or more sugar chain aspartame is contained

-12- 1324607 (7)

Smurnm^i and/or a compound having a sugar residue as described in any one of the above (1) to (3). a manufacturing method, wherein the fat-soluble protective group is a mercapto oxime oxycarbonyl (Fmoc) group;

(5) The method for producing a sugar chain aspartame derivative according to any one of the above (1), wherein the step (a) includes sialic acid at a non-reducing end A sugar chain aspartate contained in a mixture of one or more sugar chain aspartames of a residue is introduced into an Fmoc group, and a benzyl group is introduced into a sialic acid residue to obtain a sugar. (6) A method for producing a sugar chain aspartame, which comprises: (a) a step of obtaining a mixture of sugar chain aspartame derivatives, which comprises a fat-soluble protecting group is introduced onto the sugar chain aspartate of a mixture of one or more sugar chain aspartame;

(b) a step of isolating each of the sugar chain aspartate derivatives, which is derived from the sugar chain aspartate derivative mixture or the sugar chain aspartate derivative contained in the sugar chain aspartate derivative mixture a step of hydrolyzing to obtain a mixture, which is then subjected to chromatograph analysis; and (c) a step of obtaining a sugar chain aspartate which is a sugar chain aspartate derivative isolated in step (b) (7) The method for producing a sugar chain aspartame according to the above (6), which further comprises: (b') using a sugar hydrolyzate to hydrolyze the sugar chain separated in the step (b) Steps of aspartame derivatives; and/or -13- 1324607 _ (8) Explain the procedure (c'), that is, the sugar chain aspartate obtained in (c), using glycol hydrolysis The method for producing a sugar chain aspartame described in the above (6) or (7), which comprises a mixture of one or more sugar chain aspartame, Contains

-14- 1324607 (9) 丨 invention said that: and / or a compound having one or more sugar residues in the compound; (9) as described in any one of the above (6) to (8) a method for producing a sugar chain aspartame, wherein the fat-soluble protecting group is a Fmoc group;

(10) The method for producing a sugar chain aspartame described in any one of the above (6), wherein the step (a) includes a sialic acid residue on the non-reducing end. a sugar chain aspartate contained in a mixture of one or more sugar chain aspartame, which is introduced into a Fmoc group, and a thiol group is introduced on the sialic acid residue to obtain a sugar chain A step of producing a glycoside derivative mixture; (11) A method for producing a sugar chain, comprising: (a) a step of obtaining a mixture of sugar chain aspartame derivatives, which comprises containing one or more sugars a sugar-soluble protective group is added to the sugar chain aspartate of a mixture of aspartame;

(b) a step of isolating each of the sugar chain aspartate derivatives, which is derived from the sugar chain aspartate derivative mixture or the sugar chain aspartate derivative contained in the sugar chain aspartate derivative mixture Hydrolyzed to obtain a mixture, which is then subjected to chromatograph analysis; (c) a step of obtaining a sugar chain aspartate which is obtained by removing the sugar chain aspartate derivative isolated in step (b) a protecting group; and (d) a step of obtaining a sugar chain, wherein the aspartic acid residue of the sugar chain aspartate obtained in the step (c) is removed; (12) the sugar chain as described in the above (11) The manufacturing method further comprising: (b') a step of hydrolyzing the sugar chain aspartate derivative separated by the step (b); and/or -15 - 1324607 _ (10) The step C), that is, the step of hydrolyzing the sugar chain aspartate obtained by (C) using glycolytic enzyme; and/or (dj step, that is, the sugar obtained by (d) a chain, a step of hydrolyzing using a sugar hydrolyzing enzyme; (13) a method of producing a sugar chain as described in the above (11) or (12) The method, which,

-16- 1324607 (11) Pages and/or a compound having one or more sugar residues in the compound; (14) The production of a sugar chain as described in any one of the above (11) to (13) The method for producing a sugar chain according to any one of the above (11), wherein the step (a) is a method for producing a sugar chain according to any one of the above (11) to (13) Containing saliva on the non-reducing end

The oligosaccharide group is added to the sugar chain aspartate contained in a mixture of one or more sugar chain aspartames of an acid residue, and a benzyl group is introduced into the sialic acid residue to obtain a benzyl group. a step of a mixture of sugar chain aspartame derivatives; (1 6) a sugar chain aspartate derivative having the general formula: R 1

(where R1 and R2 are Η,

> -17· 1324607 (12)

j invention description page

It can be the same or different, however,

(except when R1 and R2 are both φ); (1 7) - a sugar chain aspartame derivative, which has the general formula:

R X

RY (in the formula, Rx and RY, one of them is 1324607

OH

• 19· 1324607 (14) 丨Inventive Notes (18)—A sugar chain aspartate, which has a general formula

, which may be the same or different, but R3 and R4 are simultaneously • 20-1324607 (15) I invention description R1

In addition to the case); and (19) a sugar chain, which has the general formula:

-21 1324607 (16)

-22- 1324607 (17)

, or one of R5 or R6 is Η,

At the same time, the other is except for the case).

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a view showing the construction of a population of the sugar chain aspartate derivatives obtained by the present invention. Fig. 2 is a view showing the structure of a group of the sugar chain aspartate derivatives obtained by the present invention. Fig. 3 is a view showing the structure of a population of the sugar chain aspartate obtained by the present invention.

Fig. 4 is a view showing the structure of a population of the sugar chain aspartate obtained by the present invention. Figure 5 is a diagram showing the construction of a population of sugar chains obtained by the present invention. Figure 6 is a diagram showing the construction of a population of sugar chains obtained by the present invention. Fig. 7 is a view showing an example of the steps in the method for producing the sugar chain aspartame derivative obtained by the present invention. Fig. 8 is a view showing an example of a conversion step of a sugar chain aspartame derivative using various sugar hydrolyzing enzymes. Fig. 9 is a view showing an example of a conversion step of a sugar chain aspartame derivative using various sugar hydrolyzing enzymes. -23 - 1324607 _ (18) 陋 两 ] ] ] ] 图 图 图 图 图 图 图 图 图 图 图 图 图 图 图 图 图 图 图 图 图 图 图 图 图 图 图 图 图 图 图 图 。 Fig. 11 is a view showing an example of a conversion step of a sugar chain aspartame derivative using various sugar hydrolyzing enzymes. Fig. 12 is a view showing an example of a step of removing a protecting group (Fmoc group) from a sugar chain aspartate derivative and a step of removing an aspartic acid residue from a sugar chain aspartame. Best mode for carrying out the invention

The method for producing a sugar chain aspartame derivative of the present invention, for example, is characterized in that a sugar chain aspartate which is derived from a natural sugar protein is desirably bonded by aspartate a sugar chain, a mixture of the obtained sugar chain aspartame, which is introduced into the sugar chain aspartate, and a fat-soluble protective group is introduced (bonded) thereto to obtain a sugar chain aspartate. After the mixture of amine derivatives, each sugar chain aspartate derivative is isolated from the mixture. Further, in the present specification, the term "sugar chain aspartate" refers to a sugar chain in which the aspartic acid is bonded. In addition, the so-called "aspartate-bonded sugar chain" refers to the N-acetylglucoside present at the reducing end of the acid amine group of Asparagine (Asn) in the protein's polypeptide. An amine N-glycoside-bonded sugar chain group and a sugar chain group having Man(pi-4)GlcNac as a mother nucleus. The so-called "glycosyl aspartate guanamine derivative" refers to a sugar chain aspartame which is bonded to a fat-soluble protective group on the aspartame residue. Further, in the structural formula of the compound, "AcHN" means acetamide. As described above, the sugar chain derived from the natural glycoprotein is a non-reducing sugar residue at the end of the -24-1324607 (19) having a randomly deleted sugar chain mixture. The present inventors have surprisingly found that the natural glycoprotein-derived saccharide, specifically the oligosaccharide aspartate contained in a mixture of sugar chain aspartame, is introduced by introducing a lipid. The soluble protecting group can easily separate the individual sugar chain aspartate derivatives by a conventional chromatography method using a mixture of the sugar chain aspartate derivatives into which the protecting group is introduced. Thereby, it is possible to individually prepare a large amount of a sugar chain aspartame derivative having various structures. For example, in the past, it is difficult to isolate, and the groups of compounds 2 and 6 shown in Fig. 1 or the similarly constructed sugar chain aspartate derivatives of compounds 3 and 7 shown in Fig. 2 can be separated, and These compounds can be individually and easily prepared in large quantities. Further, based on the obtained sugar chain aspartate derivative, for example, by repeating the action of the sugar hydrolyzing enzyme to remove the sugar residue, various sugar chain asparagine derivatives can be further synthesized. Thus, by introducing a fat-soluble protective group into the sugar chain aspartate to form a derivative, the individual sugar chain aspartate derivatives can be separated, and the fat-soluble protection is introduced. The fat solubility of the entire sugar chain aspartate derivative is improved. For example, the interaction with the inverse phase column which is very suitable for use can be remarkably improved, and as a result, the tax can be more sensitive. The difference reflects the structure of the sugar chain, so that individual sugar chain aspartate derivatives can be isolated. For example, the Fmoc group of a fat-soluble protecting group suitable for use in the present invention has a very high fat solubility. That is, the Fmoc-based fluorenyl skeleton has two benzene ring bonds at its central five-membered ring to form a very highly fat-soluble structure, for example, -25-13324607 13⁄4 illustrates ill (20) The octadecyl group of the ODS column, which is one of the reverse phase column, produces a very strong interaction, so that a similarly constructed sugar chain aspartate derivative can be separated. Further, according to the present invention, as described later, by removing the protective group of the obtained sugar chain aspartate derivative, various sugar chain asparagine can be obtained, and, by the obtained sugar The removal of the asparagine residue of the aspartame can also easily and in large quantities obtain various sugars. The method for producing a sugar chain aspartame derivative of the present invention, specifically, (a) a step of obtaining a mixture of sugar chain aspartame derivatives, which comprises containing one or more sugar chain aspartame a mixture of the sugar chain aspartate, introducing a fat-soluble protecting group; and (b) a step of isolating each sugar chain aspartate derivative, which is a mixture of the sugar chain aspartame derivative or The sugar chain aspartate derivative contained in the sugar chain aspartate derivative mixture is hydrolyzed to obtain a mixture, which is then subjected to chromatography analysis.

a mixture containing one or more kinds of sugar chain aspartame used in the step (a), as long as it is one or more sugar chains containing an aspartame-bonded state, and There are no special restrictions. For example, it may be a mixture containing one or more kinds of one or more linked sugar chains containing asparagine. In particular, when it is particularly preferable from the viewpoint of easy availability, it is preferred to contain one or a mixture of two or more kinds of sugar chains in which aspartic acid is bonded to the reducing end. In the present specification, the term "sugar chain" means two or more types of arbitrary monosaccharides. -26- 1324607 (21) 嗣ϋ

This mixture of sugar chain aspartame can be obtained by a known method, and is preferably obtained from a natural raw material such as milk, bovine-derived fetuin or egg-derived glycoprotein and/or glycopeptide. In the mixture, for example, a proteolytic enzyme (for example, "Pro" enzyme (manufactured by Wako Pure Chemical Co., Ltd.), "Akchi" enzyme-Ε (manufactured by Scientific Research Co., Ltd.), and a general carboxyl peptide enzyme can be added. An enzyme such as an amino-based peptide enzyme, which is subjected to a reaction under a conventional reaction condition, and which cuts off the peptide portion, can be used as a reaction liquid after the reaction, or a reaction chain other than the sugar chain aspartame. The components can be obtained by a known method (for example, a gel filtration column, an ion exchange column, and the like, and various high-performance liquid chromatography (HPLC) purification methods). Based on the easiness of production, a conventionally derived egg-derived glycopeptide [B i 〇chimicaet Biophysica Acta 1 335 (1997) p 23-32; crude purified SGP (the mixture contains protein and inorganic in egg yolk) It is desirable that the salt or the like is present and the content of the glycopeptide is from about 10 to about 80% by weight.

Further, based on the viewpoint of efficiently obtaining a sugar chain aspartame derivative having a desired sugar chain structure, it is desirable to further hydrolyze the sugar chain aspartate contained in the mixture, and to partially deposit a sugar in advance. It is preferred that the residue is cleaved. Further, the degree of the cutting is not particularly limited as long as it can be at least within the structural range of the "sugar chain" mentioned in the specification. The mixture thus obtained, for example, a mixture containing the compound 24 shown in Fig. 3 and/or a compound having one or more sugar residues in the compound is used. Further, in this case, the sugar residue has a compound which is deleted, and based on the viewpoint of maintaining the structure of the "sugar chain" mentioned in the present specification, -27- 1324607 _ (22), the sugar residue is deleted by 9 Its upper limit. For example, the compound 2 5 and 2 9 shown in FIG. 3 may be a mixture of a sugar chain aspartame containing a compound 24 (hereinafter referred to as a mixture of compounds 24), which is hydrolyzed by acid as shown below. The mixture can be efficiently obtained, and even the corresponding sugar chain aspartate derivative, that is, the compounds 2 and 6 shown in Fig. 1, can be obtained efficiently. For example, an appropriate amount of 0.1 aqueous acid solution may be added to the compound 24 mixture in an appropriate amount, and the reaction is carried out at 4 to 100 °C. At this time, on the one hand, the thin layer chromatography method (for example, using ruthenium gel 60F254 (manufactured by Merck), and ethyl acetate as a developing solvent: methanol: water = 4:4:3) is used to trace the hydrolysis. The reaction proceeds and the reaction is stopped at the maximum of the compounds 2 5 and 29. For example, it can be reacted at 25 ° C for about 5 to 10 hours, or at a temperature of 100 ° C for several minutes, and then the reaction is stopped. Ideally, the reaction can be carried out at 70 ° C. After the start of the reaction, the reaction is stopped at 35 minutes and rapidly ice-bathed. Further, the reaction can be stopped by neutralizing the reaction solution. Further, the acid is not particularly limited, and for example, an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid or trifluoroacetic acid, an organic acid, a cation exchange resin, or an insoluble solid reagent may be used. Similarly, compound 33 can be obtained from compound 24 in a very efficient manner from compound 24. For example, an appropriate amount of the aqueous acid solution may be added to the mixture of the compound 24, and it is preferred to carry out the reaction at 80 ° C. After the start of the reaction, the reaction is preferably stopped at 60 minutes. Further, it is also possible to carry out hydrolysis by an enzyme. The enzyme used at this time is suitable for glycolytic enzymes, and the endo type or exo type is either -28 - 1324607 (23) I. use. For example, in the same manner as described above, when the compound 25 and the compound 29 are obtained from the compound 24, a sialic acid hydrolyzing enzyme having a sialic acid cut off from the end can be used. The enzyme is not particularly limited as long as it has such activity, regardless of a commercially available enzyme, a novel isolated enzyme, or an enzyme produced on a genetically engineered product. The enzyme reaction can be carried out according to the conventional conditions. In this case, the reaction can be carried out by thin layer chromatography as in the above, and the reaction can be stopped at the maximum of the compounds 2 5 and 29, that is, " 5J- 〇

A mixture containing the above-obtained sugar chain asparagine was used, and a fat-soluble protecting group was introduced thereon. The protecting group is not particularly limited, and for example, a carbonate of a Fmoc group and a tributyloxycarbonyl (Boc) group, a mercapto group, an allyl group, an allyloxy carbonate group, an ethyl fluorenyl group or the like can be used. A protecting group or a amide-based protecting group. Based on the obtained sugar chain aspartate derivative, it can be used for the synthesis of a desired glycopeptide, which is preferably an Fmoc group and a Boc group, and an Fmoc group is more preferable. Fmoc base

It is particularly effective when the unstable sugar is present on the sugar chain under conditions such as allyl group which is relatively acidic. Further, the method of introducing the protecting group can be carried out according to a conventional method (for example, refer to Protecting groups in Organic Chemistry, John).

Wiley & Sons INC., New York 1991, ISBN 0-47 1 -62301-6). For example, when an Fmoc group is used, an appropriate amount of acetone may be added to a mixture containing a sugar-bonded aspartame, and then further added with a methyl group of N-succinic acid carbonate and sodium hydrogencarbonate for dissolution. The bonding reaction of the Fmoc group with the asparagine residue is carried out at 25 ° C, and Fmoc* • 29-1324607 (24) can be described as il; introduced into the sugar chain aspartate residue. According to the above procedure, a mixture of a sugar chain aspartate derivative into which a fat-soluble protective group is introduced can be obtained. Then, in the step (b), the sugar chain aspartame derivative mixture is separated into various sugar chain aspartame derivatives in a conventional color layer analysis, particularly a color separation layer analysis. Further, in this step, the sugar chain aspartate derivative mixture obtained in the above step (a) can be used as it is. However, based on the viewpoint of efficiently obtaining a sugar chain guanamine oxime derivative having a desired sugar chain structure, the sugar chain aspartate derivative contained in the mixture may be further hydrolyzed to form a part of the sugar residue. The mixture was cut off in advance and a mixture of the obtained sugar chain aspartate derivatives was used. Further, the degree of cleavage of the sugar residue can be the same as described above. Further, the hydrolysis can be the same as described above. For example, when the compounds 3 and 7 are isolated from the mixture of the compounds 2 and 6, the mixture may be administered to a hydrolysis treatment (procedure) using galactose hydrolysis. Further, the mixture can be further separated into the compounds 3 and 7 by HPLC, and various compounds of a single compound type can be obtained in a large amount. Separation of each sugar chain aspartame derivative by a chromatography method can be carried out by a combination of individual or plural layers by an appropriate and conventional chromatography method. For example, the obtained sugar chain aspartate derivative mixture can be purified by gel filtration column chromatography and purified by HPLC. The column used in HPLC is ideal for reverse phase column • 30· 1324607 (25). For example, O D S, phenyl, nitrile and anionic columns are known, such as the Mono Q column made by Pharmacia, and the Yadolo microbead column made by the company. The separation conditions and the like may be adjusted by referring to appropriate and conventional conditions. According to the above procedure, the desired individual sugar chain aspartate derivatives can be obtained from a mixture of sugar chain aspartame derivatives. An example of the steps in the method for producing the sugar chain aspartame derivative of the present invention is shown in Fig. 7.

Further, by hydrolyzing the sugar chain aspartate derivative isolated in the step (b), the sugar chain aspartate derivative having the desired sugar chain structure can be efficiently obtained [step (b')] . For example, in the separation stage of the sugar chain aspartame derivative, the type of the sugar chain aspartate derivative contained in the mixture is restricted, and the sugar chain aspartate derivative can be roughly separated, and then Hydrolysis, for example, by hydrolysis using a glycohydrolyzing enzyme, can efficiently obtain a sugar chain aspartate derivative having a desired sugar chain structure. Further, the hydrolysis can be carried out in the same manner as described above. In particular, in view of the fact that a sugar chain aspartame derivative having a desired sugar chain structure is more efficiently obtained, it is preferred that the sugar residue is cleaved by using a clear sugar hydrolyzing enzyme. For example, compounds 3 and 7 of compounds 2 and 6 which remove galactose residues are converted (step) (Fig. 8). Compounds 2 and 6 can be dissolved in a buffer (for example, phosphate buffer solution, acetate buffer) In a solution, a good buffer solution, or the like, a galactose hydrolase is used according to a conventional condition, and a galactose residue is cleaved. Further, the compounds 2 and 6 can be separated individually even if they contain a mixture type. The galactose hydrolyzing enzyme of -31 - 1324607 (26) used in this reaction is preferably a commercially available Exo-type enzyme. Further, as long as it has the same activity, it may be an enzyme that has just been isolated, and may be a genetically engineered one. Further, in the same manner as described above, the reaction liquid obtained after the reaction (a mixture of the sugar chain aspartate derivatives in which the sugar residue is cleaved) can be supplied to the chromatographic analysis, and the sugar chain aspartate can be obtained. Amidoxime derivatives are also acceptable. For example, it is desirable to carry out the separation by HPLC (ODS column, developing solvent: 50 m aqueous solution of acetic acid: acetonitrile = 8 2 : 15).

Compounds 3 and 7 of compounds 3 and 7 are removed by a ruthenium-acetamidine glucosamine residue, and the conversion (step) (Fig. 8) can dissolve compounds 3 and 7 in a buffer (for example, phosphate buffer solution, acetic acid) In a buffer solution, a good buffer solution, or the like, ruthenium-acetamidine glucosamine hydrolase is used according to a conventional condition, and a cleavage reaction of a hydrazine-acetamidine glucosamine residue is carried out. Further, Ν-acetylhexosamine hydrolyzing enzyme can also be used. Further, the compounds 3 and 7 can be separated individually even if they contain a mixed form. Each of the enzymes used in the reaction is preferably a commercially available Exo-type enzyme. Further, as long as it has the same activity, it can be an enzyme that has just been isolated, and can also be produced by genetic engineering. Further, in the same manner as described above, the reaction liquid obtained after the reaction (a mixture of the sugar chain aspartate derivatives in which the sugar residue is cleaved) can be supplied to the chromatographic analysis, and the sugar chain aspartate can be obtained. Amidoxime derivatives are also acceptable. For example, it is desirable to carry out the separation by HPLC (ODS column, developing solvent: 50 m aqueous ammonium acetate solution: acetonitrile = 6 5 : 3 5 or 50 m aqueous ammonium acetate solution: acetonitrile = 82: 15). Compounds 5 and 9 of compounds 4 and 8 with mannose residues removed, which are converted to -32-(27) (step) (Fig. 8). Compounds 4 and 8 can be dissolved in "flush solution, acetic acid buffer solution, good =: For example, the conditions of the wall are known to use the mannose hydrolyzing enzyme, the liquid, etc., and the cleavage of the glucosinolate residue is obtained. Further, the compounds 4 and 8, which are Also, π ^ 1 contains the mixture type to be separated individually. The hydrolysis used in the reaction is as long as it is the same as the genetically engineered enzymes to use commercially available ( The scorpion-type enzyme is ideal. 'The team is the two choices & I, - 户 纟 纟 纟 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再 再A mixture of aspartame organisms is added to the chromatographic analysis, and each sugar chain is obtained as long as it has the same activity, and may be an enzyme that has just been isolated, and a guanamine derivative may also be used. For example, H pl C (0 DS column, the solvent can be appropriately mixed and used as 10-200 mM ammonium acetate, etc. A solution of lytic acid and acetonitrile or a water-soluble organic solvent such as ethyl alcohol, methanol, butanol or propanol. In this case, 5 0 m Μ ammonium acetate: acetonitrile = 8 2 : 1 8 Sword) is ideal for separation. Compound ίο, which removes the galactose residue, is converted (step)

(Fig. 9) Compound 1 can be dissolved in a buffer (for example, a phosphate buffer solution, an acetic acid buffer solution, a good buffer solution, etc.), and galactose hydrolyzate is used to perform galactose residue digestion according to a conventional condition. Obtained by the reaction. In the reaction solution obtained after the reaction (mixture of sugar chain aspartame amine derivative in which the sugar residue is cleaved), the separation of the sugar chain aspartate derivative is exemplified by HPLC (ODS column) It is desirable to carry out the separation in a solvent of 50 mM aqueous ammonium acetate: acetonitrile = 85: 15). Further, Compound 11 can be further hydrolyzed using any of the sugar hydrolyzing enzymes, and is converted into various sugar chain scorpio 1 stilbenamine derivatives (for example, "," And 13, etc.. Removal of Κί ^ (Step 12 化合物 化合物 glucosamine residue compound 11 compound 12, which is acid-buffered to dissolve 9) can dissolve compound U in a buffer (for example, a conventional condition) In a viscous acid buffer, a good buffer solution, etc., according to the use of a sugar amine residue, the glucosamine hydrolase is obtained, and the ruthenium-ethyl ruthenium residue is cleaved by a 0-cut reaction. a reaction chain (a mixture of a sugar aspartame, a sugar chain, a mixture of aspartic amine derivatives), a sugar chain

For the separation of pipe branches and exhibiting organisms, for example, HPLC (0DS separation 1 solvent 50 mM ammonium acetate aqueous solution: acetonitrile = 85: u) is preferred. In addition to the glycan (Fig. 9), the compound 13 of the compound 12 of the scaly residue can be converted, and the conversion step (step) solution, although the acid-reducing compound 12 is dissolved in the buffer (for example, the glycerin buffer solution is used for the nectar In solution, good buffer solution, etc., according to the conventional article. After the reaction: the enzyme 'cuts the mannose residue to obtain a reaction solution of the guanamine derivative ( (a mixture of sugar chain aspartic organisms in which the sugar residue is cleaved), and each sugar is separated, for example. Said, as a mountain, amidoxime and a living organism, the solvent is 50 two-body column chromatography analysis (10) S column, melon cold d horse 50 acetic acid.文录. B ride m) to separate the chemical compounds 3 and 7 into the sinister servants 3 and 7, 々 ° and 19 'the transformation (step) (Figure J 〇) can dissolve compounds 3 and 7 Buffer (for example) using saliva, acetic acid buffer solution, good buffering cold liquid acid hydrolysis enzyme, into,,,,, Yin, according to the conditions of the known, sputum residue residue of the reaction -34 · 1324607 (29) 丨[j Ming-saymi

. Further, the compounds 3 and 7 can be separated individually even if they contain a mixture type. The sialic acid hydrolyzing enzyme used in this reaction is preferably a commercially available exo-type enzyme. Further, as long as it has the same activity, it may be an enzyme that has just been isolated, and may be an author of genetic engineering. Further, in the same manner as described above, the reaction liquid obtained after the reaction (a mixture of the sugar chain aspartate derivatives in which the sugar residue is cleaved) can be supplied to the chromatographic analysis, and the sugar chain aspartate can be obtained. Amidoxime derivatives are also acceptable. For example, high-speed liquid column chromatography (ODS column, developing solvent is 50 mM ammonium acetate aqueous solution: acetonitrile = 8 5 : 15) for separation.

Compounds 15 and 19 of compounds 14 and 19 from which N-acetylglucosamine residues are removed are converted (step) (Fig. 10). Compounds 14 and 19 can be dissolved in a buffer (for example, an acid buffer solution, In an acetic acid buffer solution, a good buffer solution or the like, N-acetylglucosamine hydrolase is used according to a conventional condition, and a N-acetylglucosamine residue is subjected to a cleavage reaction. In the reaction solution obtained after the reaction (mixture of sugar chain aspartate derivatives in which the sugar residue is cleaved), each sugar chain aspartate derivative is analyzed by a high-speed liquid column chromatography layer (〇DS tube) Column, the developing solvent is 50 m Μ acetic acid aqueous solution: acetonitrile = 82: 18) Separation is ideal. The compounds 15 and 20 of the compounds 15 and 20 from which the mannose residue is removed are converted (step) (Fig. 10). The compounds 15 and 20 can be dissolved in a buffer (for example, a phosphate buffer solution, an acetic acid buffer solution, a good buffer). In the solution or the like, in the same manner as described above, a mannose hydrolyzing enzyme can be used according to a conventional condition, and a mannose residue can be obtained by a cleavage reaction of a mannose residue. The anti-35 - 1324607 _ (30) obtained after the reaction is described in the continuation of the noble liquid (a mixture of the sugar chain aspartate derivatives of the sugar residue cleaved), each sugar chain aspartame derivative It is desirable to carry out the separation by high-speed liquid chromatography (ODS column, developing solvent: 50 mM aqueous ammonium acetate: acetonitrile = 82:18). Compounds 16 and 2, which remove galactose residues, are converted (steps) (Fig. 11). Compounds 16 and 21 can be dissolved in a buffer (for example, a phosphate buffer solution). In the acetic acid buffer solution, a good buffer solution, or the like, a galactose hydrolyzate is used according to a conventional condition, and a galactose residue is subjected to a cleavage reaction. In the reaction solution obtained after the reaction (mixture of sugar chain aspartate derivatives in which the sugar residue is cleaved), the separation of each sugar chain aspartate derivative is, for example, a high-speed liquid chromatography layer. It is desirable to carry out the separation (Ο DS column, developing a solvent of 50 m Μ ammonium acetate aqueous solution: acetonitrile = 8 5 : 1 5 ) for separation. Compounds 17 and 23 of compounds 17 and 22, which have been removed from the oxime-acetylglucosamine residue, are converted (step) (Fig. 11). Compounds 17 and 22 can be dissolved in a buffer (for example, phosphate buffer solution, acetic acid) In the buffer solution, the good buffer solution, and the like, in the same manner as described above, ruthenium-acetamidine glucosamine hydrolase was used according to a conventional condition, and a cleavage reaction of hydrazine-acetamidine glucosamine residue was carried out. In the reaction solution obtained after the reaction (mixture of sugar chain aspartate derivatives in which the sugar residue is cleaved), each sugar chain aspartate derivative, for example, is a high-speed liquid column color layer. It is desirable to carry out the separation (〇DS column, developing solvent: 50 m aqueous ammonium acetate solution: acetonitrile = 8 2 : 18). Thus, after obtaining each sugar chain aspartate derivative, various sugar hydrolyzing enzymes and the like can be further used to hydrolyze the derivative, and the sugar residue of the non-reducing end of the sugar chain can be removed by removing -36-1324607 (31) For example, each of the forms of the single compound gives a variety of sugar chain aspartate derivatives having a branched structure at the end of the sugar chain. Further, various kinds of sugar hydrolyzing enzymes can be used, and by changing the order of hydrolysis and the kind thereof, more kinds of sugar chain aspartame derivatives can be produced. According to the past method, when it is desired to obtain a sugar chain aspartate derivative having a limiting sugar chain structure on an analytical scale, it takes a lot of time and expense, but according to the present invention, no special apparatus and reagents are required, as long as it is used. Conventional gel filtration column, HPLC column, and at least three kinds of glycolytic enzymes (for example, nougat hydrolase, mannose hydrolase, N-acetylglucosamine hydrolase), etc., can be about two weeks At the time, one gram of a sugar chain aspartate derivative having a desired sugar chain structure was produced. According to the following procedure, for example, when the protecting group is an F m 〇 c group, a sugar chain aspartate derivative having the following general formula can be efficiently produced. 1

R

As n-Fm〇 c

r°〇H r-Ο»

ί ΑϋΗΛ AcHH ID 2 (where R1 and R2 are H, -37- 1324607 _ (32) Months say ϋ

Except when). Such a sugar chain aspartate derivative, specifically, for example, each compound shown in Fig. 1 and Fig. 2 . Among them, in particular, Compound 1, Compounds 2 to 9, 1 1 to 2 3, 70 and VII are produced by the present invention for the first time. The scope of the invention includes the compound. Further, in the method for producing a sugar chain aspartame derivative of the present invention, an ideal aspect is to provide a method for producing a sugar chain aspartame derivative, which is -38 to 1324607

(33) Hidden SUB

Step (a) is a mixture of one or more sugar chain aspartame containing a sialic acid residue at a non-reducing end, and introducing a Fmoc group to the sugar chain aspartate contained in the mixture. At the same time, a step of introducing a benzyl group into a sialic acid residue to obtain a mixture of sugar chain aspartate derivatives is obtained. According to this production method, various types of sugar chain aspartate derivatives can be obtained in a large amount more efficiently. For example, the separation efficiency of the compounds 2 and 6 shown in Fig. 1 can be improved, and the production of the di compound can be carried out efficiently. That is, the separation of the two compounds directly and clearly from the mixture of the compounds 2 and 6 is very disadvantageous in efficiency, but in this aspect, if a thiol group is introduced on the sialic acid residue of the di compound, the following a mixture of compounds 7 6 and 7 7 ,

-39- 1324607 (34)

-40- 1324607 (35) Description of the Invention

-41 - 1324607 _ (36) Mingtong] When the above step (b) is added, the separation of the compounds 76 and 77 can be carried out relatively easily, and the two compounds can be separated, and then the benzyl group can be further described below. When it is detached, it can be obtained by efficiently separating the two compounds from the mixture of the compounds 2 and 6. The separation of the compounds 2 and 6 is quite difficult, but the separation of the compounds 7 6 and 7 7 obtained by introducing a thiol group to the sialic acid residue group is easy, because the carboxyl group of the sialic acid residue is The introduction of a high-fat-soluble sulfhydryl group is further enhanced (separation efficiency) by interaction with the hydrophobic phase interaction between the reverse phase column of HPLC (High Speed Liquid Chromatography). Therefore, it is presumed that in the separation step (b), the interaction between the column and the column which is appropriately used is remarkably enhanced, and as a result, the difference between the sugar chain structures is more acutely reflected, and the two compounds can be separated. The mixture containing the sugar chain aspartate having one or two or more kinds of sialic acid residues at the non-reducing end is one or a mixture of two or more kinds of the sugar chain aspartame having such a structure. Yes, there are no special restrictions. From the viewpoint of easy availability, it is preferred to contain one or a mixture of two or more sugar chains having a sialic acid residue at the non-reducing end and an aspartic amine at the reducing end. The mixture is preferably one containing the compound 24 shown in Fig. 3 and/or a compound having one or more sugar residues in the compound. Further, from the viewpoint of more efficiently obtaining a sugar chain aspartate derivative having a desired sugar chain structure, it is desirable to carry out the above step (b'). Further, in this aspect, the sugar chain aspartate derivative -42-1324607 (37) [13⁄4^3⁄43⁄43⁄4 is introduced into the group having the word group and the Fmoc group, and only the Fmoc group is introduced. Regarding the sialic acid residue of the thiol-introduced sugar chain aspartate, as long as it is according to a conventional method (for example, refer to Protecting groups in Organic Chemistry, John Wiley & Sons INC., New York 1991, ISBN 0-47 1-6230 1 -6)^ ^ 0 According to the above few steps, for example, the following general formula can be obtained efficiently:

RY (where Rx and K/ are one)

The other is Η, • 43-1324607 _ (38)

• Or volume l is also introduced with a thiol and Fmoc-based sugar chain aspartate derivative. Such a sugar chain aspartate derivative, specifically, the aforementioned compounds 76 and 77, and the following compound 78: -44- 1324607

(39) S Ming MSS

·45· 1324607 (40) Explain the continuation page. The sugar chain aspartate derivative obtained according to this aspect can be directly used for solid phase synthesis of a glycopeptide. a carboxyl group present on the sialic acid residue of the sugar chain aspartate derivative, which is protected by a benzyl group, compared to a sugar chain aspartame derivative having no mercapto group introduced therein The advantage is that in the solid phase synthesis of the glycopeptide, there is no side reaction in which the carboxyl group participates, and the desired glycopeptide can be obtained efficiently. Further, the present invention provides a process for producing a sugar chain aspartame which can obtain a large amount of various isolated sugar chain aspartame. The method includes the steps of: the step of producing the sugar chain aspartame derivative according to the method for producing the sugar chain aspartame derivative, and further, the obtained sugar chain aspartame The step of removing the protecting group from the amine derivative. That is, the method for producing a sugar chain aspartame of the present invention comprises the step of (a) obtaining a mixture of sugar chain aspartame derivatives, which comprises containing one or more sugar chain aspartames. a sugar-soluble protective group is introduced onto the sugar chain aspartate of a mixture of amines; (b) a step of isolating each sugar chain aspartate derivative, which is a mixture of the sugar chain #aspartame derivative Or a sugar chain aspartate derivative contained in the sugar chain aspartate derivative mixture, hydrolyzed to obtain a mixture, which is then subjected to chromatographic analysis; and (c) obtaining a sugar chain aspartate The step of removing the protecting group of the sugar chain aspartate derivative isolated in the step (b). The step (a) and the step (b) are the same as the method for producing the aforementioned sugar chain aspartate derivative, and the mixture containing one or more kinds of sugar chain aspartame is used, and the fat-soluble protection is used. The base is also the same as described above. -46- 1324607 (41) I invention descriptionϋ

In the step (c), the method of removing the protective group from the sugar chain aspartate derivative can be carried out according to a conventional one (for example, refer to Protecting groups in Organic Chemistry, John Wiley & Sons INC., New York 1991, ISBN 0-471-62301-6). For example, if the protecting group is an Fmoc group, as shown in Figure 12, can it be added to the sugar chain aspartate derivative in N,N-dimercaptodecylamine (DMF)? The morpholine is reacted to remove the Fmoc group. Further, the Boc group can also be removed by a weak acid reaction. After the protective group is removed, it can be purified by various chromatographic methods such as a gel filtration column, an ion exchange column, and the like, and a pyridine PLC separation method, as desired, to obtain a sugar chain aspartate also "5J&quot Further, in the same manner as the method for producing the sugar chain aspartate derivative, the method for producing a sugar chain aspartame of the present invention provides a method for producing a sugar chain aspartame derivative. And the step (a) is carried out by using a mixture of one or more sugar chain aspartame containing a sialic acid residue at a non-reducing end, and introducing the sugar chain asparagine in the mixture. The Fmoc group is a step in which a thiol group is introduced into a sialic acid residue to obtain a mixture of sugar chain aspartate derivatives. In this aspect, step (c) removes the thiol group in addition to the Fmoc group. The removal of the thiol group can be carried out according to a conventional method (for example, refer to Protecting groups in Organic Chemistry, John Wiley & Sons INC.,

New York 1 99 1, ISBN 0-47 Bu 623 (Π-6). Further, based on the efficient obtaining of the sugar chain aspartate having the desired sugar chain structure, it is desirable to hydrolyze the sugar chain aspartate-47- 1324607 _ (42) amine derivative isolated in the step (b). And/or hydrolyzing the sugar chain aspartate obtained in step (c). Further, the hydrolysis can be carried out in the same manner as described above. The viewpoint of obtaining the sugar chain aspartate having a desired sugar chain structure more efficiently is preferably carried out by using a glycolysis enzyme for the hydrolysis [step (b') and/or step (c')]. According to the above steps, for example, a sugar chain aspartate having the following general formula can be efficiently obtained:

Asn (where R3 and R4 are Η

•48· 1324607 (43)

Except in the case of). Such a sugar chain aspartate, specifically, for example, each compound shown in Fig. 3 and Fig. 4 . Among them, in particular, compounds 25 to 32, 34 to 46, 72 and 73 are the first manufacturers of the present invention. The scope of the invention encompasses such compounds. Further, the present invention provides a method for producing a sugar chain which can obtain a large number of separated sugar chains in a large amount. The method further comprises, in addition to the method for producing the sugar chain aspartame, after the step of producing the sugar chain aspartame, further comprising removing the sugar chain aspartate residue from the obtained sugar chain aspartate. The basis of the steps. That is, the method for producing a sugar chain of the present invention comprises: -49- 1324607 _ (44) (a) a step of obtaining a mixture of sugar chain aspartame derivatives, which comprises containing one or more sugars a sugar-soluble protective group is introduced on the sugar chain aspartate of a mixture of aspartame; (b) a step of isolating each sugar chain aspartate derivative, which is the sugar chain aspartate a derivative of the derivative or a sugar chain aspartate derivative contained in the mixture of the sugar chain aspartate derivative, which is hydrolyzed to obtain a mixture, which is then subjected to chromatographic analysis; (c) obtaining a sugar chain aspartate a step of removing guanamine, which is a step of removing the protecting group of the #glycan aspartate derivative isolated in the step (b); and (d) obtaining a sugar chain, which is obtained by the step (c) The aspartate residue of the sugar chain aspartate is removed. Steps (a) to (c) are the same as the method for producing the aforementioned sugar chain aspartate derivative, and a mixture containing one or more kinds of sugar chain aspartate and a fat-soluble protective group are used. , also the same as before. Further, in the method for producing a sugar chain of the present invention, it is preferred to provide a method for producing a sugar chain aspartame, wherein the step (a) is carried out using a sialic acid residue at a non-reducing end. a mixture of one or more sugar chain aspartame, introducing a Fmoc group to the sugar chain aspartate contained in the mixture, and introducing a thiol group to the sialic acid residue to obtain a sugar chain The step of the mixture of the benzamine derivative. In this aspect, step (c) removes the word base in addition to the Fmoc group. The removal of the leather base can be carried out according to the aforementioned method. In the step (d), the aspartame residue is removed from the sugar chain aspartame, and it can be carried out according to a conventional method. For example, as shown in Figure 12, the model-50- 1324607 (45) "-------------------------------------------------------------------------------------------------------- A sugar chain can be obtained by a guanamine residue. Further, after the sugar chain aspartate is heated and refluxed with an aqueous alkaline solution, the sugar chain can be obtained by removing the aspartate residue by acetylation. After the glycidyl residue is purified, it can be purified by a suitable and conventional method such as a gel filtration column, various color layer analysis methods using an ion exchange resin, and the like, and HPLC separation.

Further, in the same manner as described above, based on the viewpoint of efficiently obtaining a sugar chain of a desired sugar chain structure, the sugar chain aspartate derivative separated in the step (b) is hydrolyzed, and/or the step (c) is used. It is preferred that the obtained sugar chain aspartate is hydrolyzed and/or the sugar chain obtained in the step (d) is hydrolyzed. Further, the hydrolysis can be carried out in the same manner as described above. The viewpoint of obtaining a sugar chain having a desired sugar chain structure more efficiently by using a glycolysis enzyme for hydrolysis [step (b') and/or step (c') and/or step (d')] For the better. According to the above steps, for example, a sugar chain having the following general formula can be efficiently obtained:

(wherein, R5 and R6 are Η, -51 - 1324607 _ (46) [Inventive description page

•52· 1324607 Description of the Invention (47)

Except in the case of). Such a sugar chain aspartame is specifically, for example, each compound shown in Fig. 5 and Fig. 6. Among them, in particular, compounds 48 to 5 5, 57 to 69, 74 and 7 5 are the first manufacturers of the present invention. The scope of the invention encompasses such compounds. As described above, according to the present invention, a sugar chain aspartame derivative, a sugar chain aspartate, and a sugar chain having a desired sugar chain structure can be efficiently produced at a high cost (hereinafter, there will be The merger of the three is called the sugar chain). This sugar chain is very useful in fields such as pharmaceutical development. For example, one of its application examples is a vaccine for cancer. It is known that if a cell becomes cancerous, it will have a sugar chain that is not present in the body. Further, it is known that when the sugar chain is chemically synthesized and administered as a vaccine to an individual, the proliferation of cancer can be controlled. Thus, as long as the desired sugar chain can be produced according to the present invention, it is possible to treat cancer with the synthesis of an effective vaccine by using -53- 1324607 _ (48) [S. Further, a novel vaccine can be synthesized by combining a sugar chain obtained by the present invention with a chemical reaction and a sugar transferase reaction to form a derivative with a new sugar residue. Further, for example, erythropoietin (EPO) can be used as a therapeutic drug for anemia by virtue of its red blood cell proliferation ability, but it is known that it is not active if it is not bonded to a sugar chain. In this way, since the protein can express its physiological activity by the linkage of the sugar chain, for example, in the coliform expression system in which the sugar chain cannot be bonded, only a large amount of protein is produced, and then the desired sugar is obtained. When the chain structure is introduced and the oxime chain produced by the present invention is introduced, the physiological activity can be expressed, and when a sugar chain having various sugar chain structures and produced by the present invention is introduced into any protein, It is possible to synthesize novel glycoproteins having novel physiological activities. Further, the sugar chain present on the natural glycoprotein can be imparted with novel physiological activity by being substituted with the sugar chain produced by the present invention. The sugar chain possessed by the glycoprotein is substituted for the sugar chain obtained by the invention, for example, P. Sears and C. Η. Wong, Science, 200 1, vol 291, p 2 3 44 - 2 3 5 0 The method described. That is, when the glycoprotein is treated with /3 -N-acetylglucosaminase (Endo-H), it can cause an N-acetylglucosamine on the surface of the aspartic acid residue on the protein surface. The state of the residue. Then, using the /5-N-acetylglucosamine enzyme (Endo-M), the sugar chain aspartate (for example, each compound of FIG. 3 and FIG. 4) obtained by the present invention can be used. The desired sugar chain is bonded to the aforementioned N-acetylglucosamine residue. Further, if N-acetyl glucosamine is bonded to the tRNA, it is also possible to use a coliform such as -54-1324607 (49) to explain that the expression of the ruthenium is synthesized by synthesizing a glycoprotein having N-acetyl glucosamine. The desired sugar chain in the sugar chain aspartate obtained by the present invention is introduced using Endo-M'. Further, when glycoprotein is used as a therapeutic drug, the problem is that the metabolism of the glycoprotein to be administered is too fast. This is because the sialic acid present at the end of the sugar chain of the glycoprotein is immediately metabolized by the liver when it is removed in the living body. Therefore, it is necessary to administer a certain amount of the glycoprotein. Therefore, according to the present invention, the sialic acid which is difficult to remove at the end of the sugar chain is recombined into the sugar chain, and when the sugar chain is introduced by Endo-M on the target protein, it is possible to reduce the administered sugar. The amount of glycoprotein. Hereinafter, the invention will be specifically described by way of examples, but the invention is not limited to the examples. The structural formulae and numbering of each compound are shown in Figs. 1 to 6 . Further, the conditions of the iH-NMR measurement were as follows: in Examples 1 to 7, the HOD was 4.8 ppm at 30 ° C, and 30 in Examples 8 to 45. (The methyl group of the acetone is 2.225 ppm' and the HOD is 4.718 ppm. Further, the compound obtained by removing the Fmoc group is obtained by measuring 50 mM # of ammonium hydrogencarbonate coexisting in the measurement solvent. Example 1 Synthesis of Compound 2 4 The crude purified SGP (sialyl glycopeptide) from the egg was dissolved in Tris·hydrochloric acid•calcium chloride buffer solution (TRIZMA BASE 0.〇5mol/liter, chlorine) Calcium 0_01 Mohr / liter, pH 7.5) 100 ml. Add sodium azide 58 mg (772 μηιοί) and actinase-E 526 mg (manufactured by Scientific Research Co., Ltd.) at 37 ° C After standing for 65 hours, add 263 -55 - 1324607 _ (50) 13⁄43⁄43⁄43⁄4⁄4 mg of actin-E, and then stand at 37 ° C for 24 hours. This solution will freeze after drying. The substance was purified twice by gel filtration column chromatography (Sephadex G-25, 2.5 φ χ 1 π, developing solvent water, flow rate: 1.0 ml/min) to obtain the target compound of 1.3 g (555 μηιοί) as shown in Fig. 3. 24. The sugar chain structure contained in the SGP is as follows.

-56- 1324607 (51) I twisted 13⁄4 alum page

Further, the physical properties of the obtained Compound 24 are as follows. 'H-NMR (D2〇, 30°C) 5.15 (1H, s, Man4-H,); 5.06 (1H, d, GlcNAcl-H,), 4.95 •57· 1324607 (52) (1H, s, Man4 '-H1), 4.82 (1H, s, Man3-Hi), 4.69 (1H, d, GUNAC2-HO, 4.67 (2H, d, GlcNAc5, S'-HO, 4.53 (2H, d, Gal6, 6'- H,)5 4.3 4 ( 1 H, d, Man 3), 4.2 7 (1 H,d,

Man4'-H2)5 4.19 (1H, d, Man4-H2), 3.03 (1H, dd, Asn-β CH), 3.00 (1H, dd, Asn-β CH), 2.76 (2H, dd, NeuAc7, 7 '-H3eq), 2.15 (18H, sx6, -Ac), 1.79 (2H, dd, NeuAc7, 7'-H3ax) Example 2 Synthesis of Compounds 1, 2, 6 and 10 The compound obtained in Example 1 24 (609 mg, 261 μηιοί) was dissolved in 20.7 ml of water, and further 3.8 ml of 0.1 guanidine hydrochloride was added. This solution was taken at 70. (: After heating for 35 minutes, it was rapidly cooled, and saturated sodium hydrogencarbonate was added to make pH 7. After lyophilization, the residue was subjected to a gel filtration chromatography column (Sephadex G-25, 2.5 φ lm, developing solvent as water, Purification was carried out at a flow rate of 1.0 ml/min. to obtain a mixture of 5 34 mg of compound 24, compound 2 5, 2 9 and compound 3 as shown in Fig. 3. Do not separate the four components, And proceed to the following steps.

Further, the physical properties of the obtained sugar chain mixture are as follows. H-NMR (D20, 30 ° C) 5.13 (s, Man4-H,), 5.12 (s, Man4-H〇, 5.01 (d, GlcNAcl-H〇, 4.94 (s, Man4'-H,), 4.93 (s, Man4'-H,), 4.82 (s,

Man3-H!), 4.60 (d, GlcNAc2-H,), 4.58 (d, GlcNAc5, 5'-Hi), 4.47 (dd, Gal6, 6'-H,), 4.44 (d, Gal6, ό'- ΗΟ, 4.24 (d5 Man3-H2), 4.19 (d, Man4'-H2), 4.11 (d, Man4-H2), 2.97 (bdd, Asn-β CH), 2.72 (dd, NeuAc7-H3eq, NeuAc7-H3eq ), -58- 1324607 (53) Description of the Invention Page 2.64 (bdd, Asn-β CH), 2.15 (Sx5,-Ac), 1.79 (dd,

NeuAc7-H3ax, NeuAc7'-H3ax) The obtained sugar chain mixture 4 2 9 mg was dissolved in acetone 6.3 ml and water 11-2 ml. 9-Fluoroindenyl-N-boroic acid decanoate (155.7 mg, 461.7 μπιοί) and sodium hydrogencarbonate (80.4 mg, 957 μηηοΐ) were added thereto, and stirred at room temperature for two hours. The solution was purged with acetone in an evaporator. The residual solution was purified by gel filtration through a column (Sephadex G-25, 2.5 φ X 1 m, developing solvent water, flow rate 1. 〇 ml/min). And a mixture of 3,9 mg of the compound 2, 6 and the compound 1 was obtained as shown in FIG. The mixture was purified by HPLC (ODS column, developing solvent: 50 mM aqueous solution of ammonium acetate: acetonitrile = 65: 35, 2.0 φ X 25 cm, flow rate: 3 ml/min), respectively, and the dissolution time was respectively, Compound 1 After 5 minutes, the mixture of compounds 2 and 6 was after 6 7 minutes; compound 10 was after 9 3 minutes. The fractions were separated and lyophilized, and then purified by a gel filtration chromatography column (Sephadex G-25, 2.5 φ X 30 cm, developing solvent water, flow rate: 1.0 ml/min) to obtain the purpose. Compounds 2 and 6 were mixed with 150 mg. Further, the physical properties of the obtained compound 1' are as follows. 'H-NMR (D20, 30 ° C) 7.99 (2H, d, Fmoc), 7.79 (2H, d, Fmoc), 7.55 (4H, m, Fmoc), 5.15 (1H, s, Man4-Hi). 5.06 (1H, d, GlcNAcl-Hi), 4.95 (1H, s, Man4'-H,), 4.82 (1H, s, ManS-HO, 4.69 (1H, d, GlcNAc2-H!), 4.67 (2H, d , GlcNAc5, 5'-H,), 4.53 (2H, d, Gal6, 6'-H,), 4.34 (1H, d, Man3-H2), 4.27 (1H, d, -59- 1324607 (54) 13⁄43⁄43⁄43⁄4

Man4'-H2), 4.19 (1H, d, Man4-H2), 3.03 (1H, bdd, Asn-β CH), 3.00 (1H, bdd, Asn-β CH), 2.76 (2H, dd, NeuAc7, 7 '-H3eq), 2.15 (18H, Sx6, -Ac), 1.79 (2H, dd, NeuAc7, 7'-H3ax) ; HRMS C] 〇3H154N8Na066 [M + Na + ] Theoretical value 2581.8838, found 2581.8821° The physical properties of the obtained Compounds 2 and 6 are as follows. *H-NMR (D2〇, 30°C)

7.99 (d, Fmoc), 7.79 (d, Fmoc), 7.55 (m, Fmoc), 5.14 (s, Man4-H,), 5.12 (s, Man4-H), 5.00 (d, GlcNAcl-H,), 4.94 (s, Man4'-H,), 4.93 (s, Man4'-H1), 4.82 (s, ManS-Hj), 4.60 (d, GlcNAc2-H,), 4.58 (d, GlcNAc5s 5'-H, ), 4.46 (dd, Gal6, ό'-ΗΟ, 4.44 (d, Gal6, ό'-ΗΟ, 4.24 (d, Man3-H2), 4.19 (d, Man4'-H2), 4.11 (d, Man4-H2 ), 2.97 (bdd, Asn-β CH), 2.72 (dd, NeuAc7-H3eq, NeuAc7-H3eq), 2.64 (bdd, Asn-β CH), 2.15 (Sx5, -Ac), 1.79 (dd, NeuAc7-H3ax ,

NeuAc7'-H3ax) Further, the obtained compound 10 has the following physical properties. 'H-NMR (D20, 30 ° C) 7·99 (2H, d, Fmoc), 7.79 (2H, d, Fmoc), 7.55 (4H, m, Fmoc), 5.12 (1H, s, Man4-H, ), 5.06 (1H, d, GlcNAcl-H,), 4.93 (1H, s, Man4'-H,)5 4.82 ( 1 H, s, Man3-Hi), 4.69 (1H, d, GlcNAc2-H,) , 4.67 (2H, d, GlcNAc5, 5'-H,), 4.53 (2H, d, Gal6, 6'-H,), 4.34 (1H, d, Man3-H2), 4,27 (1H, d, Man4'-H2), 4.19 (1H, d, Man4-H2), 3.03 (1H, bdd, Asn-β -60· 1324607 (55) I invention description page CH), 3.00 (1H, bdd, Asn-β CH), 2.15 (12H, s&gt;&lt;45-Ac); HRMS C81H12 〇N6Na05 〇 [M + Na + ] calc. 1999.6930, found </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> Example 3 Synthesis of Compounds 3 and 7 a mixture of compounds 2 and 6 (2 2 4 mg, 9 7 μιηοΐ) and bovine serum albumin 24 housek'; trough in HEPES buffer solution (50 mM, pH 6.0) in 22 ml 'further added again by DiPloc〇 Ccus pneumoniae-derived point-galactosidase (1.35 U). Place ghai solution at 37. (: After standing for 15 hours, freeze-dry. The residual material was again HpLC (ODS column, 2.0 φ X 2 5 cm, developing solvent was 5 mM mM ammonium acetate aqueous solution: acetonitrile = 8 5 : 15 5, flow rate was 3 Purification was carried out in dl/min. The dissolution time was as follows: Compound 3 shown in Figure 2 was 129 minutes later; Compound 7 was after 134 minutes. Each of them was subjected to lyophilization and then to HPLC (ODS tube). Column, 2.0 φ X 25 cm, developing solvent for the first 15 minutes as water, 16 minutes to 30 minutes for water: acetonitrile (capacity ratio 10: 〇 to 85: 15, 31 minutes to 45 minutes for water: acetonitrile = 85 : a gradient of 15 to 80: 20, a flow rate of 3.0 ml/min), desalting, and the desired compound 3 was 81 mg, and the compound 7 was 75 mg. The physical property of the obtained compound 3 The system is as follows: 'H-NMR (D20, 30 ° C) 7-99 (2H, d, Fmoc), 7.79 (2HS d, Fmoc), 7.55 (4H, m, Fmoc), 5.15 (1H, S, Man4-Hi), 5.06 (1H, d, GlcNAcl-H)), 4.95 (1H, s, Man4'-Hi), 4.82 (1H, s, Man3-H!), 4.69 (1H, d, G1cNAc2-H ,)5 4.67 (2H, d, GlcNAc5, 5'-H,), 4.53 (1H, 1324607 _ (56) i invention description _ 1 d, Gal6'-H,) 5 4.3 4 ( 1 H, d, M an 3-H 2), 4 · 2 7 (1 H, d,

Man4'-H2), 4.19 (1H, d, Man4-H2), 2.97 (1H, bdd,

Asn-PCH), 2.76 (1H, dd5 NeuAc7'-H3eq)s 2.61 (1H, bdd, Asn-β CH), 2.15 (15H, sx5, -Ac), 1.79 (1H, dd,

</ RTI> <RTI ID=0.0></RTI> </ RTI> </ RTI> <RTI ID=0.0></RTI> </ RTI> </ RTI> <RTIgt; Further, the physical properties of the obtained Compound 7 are as follows. 'H-NMR (D2〇, 30°C) • 7.99 (2H,d,Fmoc), 7.79 (2H,d,Fmoc),7.55 (4H,m,

Fmoc), 5.15 (1H, S, 5.06 (1H, d, GlcNAcl-D, 4.95 (1H, s, Man4'-H1), 4.82 (1H, s, Man3-H,), 4.69 (1H, d, GlcNAc2 -Kh), 4·67 (2H, d, GlcNAc5, 5'-!!), 4.53 (1H, d, Gal6-Hi), 4.34 (1H, d, Man3-H2), 4.27 (1H, d5

Man4'-H2), 4.19 (1H, d, Man4-H2), 2.97 (1H, bdd,

Asn-pCH), 2.76 (1H, dd, NeuAc7-H3eq), 2.60 (ih, bdd, Asn-PCH), 2.15 (1 5H, sx5, -Ac), 1. 7 9 (1 h, dd, # NeuAc7 HRMS C86H125N7Na3 〇 53 [M + Na + ] calc. 2172.6995, found 2172.7084. Example 4 Synthesis of Compounds 4 and 8 A mixture of Compounds 3 and 7 obtained in Example 3 (90 mg, 47.3 μιηοΐ) was not separately isolated and dissolved in HEPES buffer solution (SOmM' together with 8 mg of bovine serum albumin. In a pH of 6.1 ml, 2.8 8 U was further added to the cold-glucosamine enzyme (manufactured by Sigma-Delci) derived from bovine kidney. The solution was allowed to stand at 37 ° C for 18 hours, then 'freeze-dried • • 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 62 &gt; 4 25 Cm development solvent for purification, the respective dissolution time is &quot;IL speed of 3 ml / min) minutes; compound 8 after 127 minutes. After the material 4 is 1 1 7 dry, and then HPLC (ODS tube 杈, 'each knife is taken and frozen for the first 15 minutes for water, 16 minutes to ^ 2 5 em, the solvent is developed 〇 minutes for water: B gambling = 10· 〇 to 85:15, 31 minutes to 45 minutes is ^ 10-0. B guess = 8 5 : 1 5 to 8 0 . 2 0 gradient, flow rate is 3 ml / min) feed, '20 desalination The target compound 4 was obtained as 40 mg, and the compound 8 was a and J horse was 37 mg. Further, the obtained compound 4 has the following team and physical properties as shown below. !H-NMR (D2〇, 30°C) 8.01 (2H, d, Fmoc), 7.80 (2H ar , d, Fmoc), 7.56 (4H, m, Fmoc), 5.22 (1H, s, Manm!), 5 〇8 (1H, d, GlcNAcl Hi), 4.94 (1H, s, Manr-H!), 4.84 (1H, s, ManS-H., 4.69 (1H, d, GlcNAc2-Hi), 4.67 (1H, d, GlcNAcS-Hj), 4.55 (1H, d, Gal6-Hi), 4.33 (1H, dd, Man3-H2), 4.20 (1H, dd, Man4-H2), 4.15 (1H, dds Man4'-H2) , 2.97 (1H, bdd, Asn-β CH), 2.76 (2H, dd, NeuAc7, 7'-Η3ες), 2.62 (1H, bdd, Asn-β CH), 2.15 (12H, sx4,-Ac), 1.79 (2H, dd, NeuAc7, 7'-H3ax); HRMS C78H1]4N6NaO48 [M + Na + ] Theory 1 925.65 62 ' Measured value 1925. 6 5 3 9 ° Further, the obtained compound 8, physical properties e 'H-NMR (D2〇, 30°c) 7.99 (2H, d, Fmoc), 7.79 (2H, d, Fmoc), 7.55 (4H, m, -63 - 1324607 (58)

Fmoc), 5.15 (1H, S, Man4-H,), 5.06 (1H, d5 GlcNAcl-HO, 4.95 (1H, s, Man4'-Hi), 4.82 (1H, s, Man3-H,), 4.69 ( 1H, d, G1cNAc2-H,)5 4.67 (2H, d, GlcNAc5, S'-H,), 4.53 (2H, d, Gal6, ό'-ΗΟ, 4.34 (1H? d, Man3-H2); 4.27 (1H, d, Man4'-H2), 2.97 (1H, bdd, Asn-β CH2), 2.76 (1H, dd, NeuAc7'-H3eq), 2.61 (1H, bdd, Asn-β CH2), 2.15 (12H , s^4, _Ac), 1.79 (1H, dd, NeuAc7'-H3ax); HRMS C78H114N6Na048 [M + Na + ] Theory 1 925.65 62, found 1 925.6 5 3 3 • Synthesis of Example 5 Compound 5 Compound 4 obtained in Example 4 (30 mg, 473 μηιοί) and 3 mg of bovine serum albumin were dissolved in 6 ml of HEPES buffer solution (50 mM, pH 6.0), and further added with α-derived from Jack bean. Mannosidase 10 U. The solution was allowed to stand at 37 ° C for 21 hours, then lyophilized, and then HPLC (ODS column '2.0 &lt; () x 25 cm, developing solvent for the first 15 minutes for water, 16 Minutes to 30 minutes for water: acetonitrile = 10: 〇 to 85: 15, 31 minutes to 45 minutes for water: acetonitrile = 85: 15 to 80: 20 gradient, flow rate ® 3 ml/min) Purification, and the compound 5 shown in Fig. 1 was obtained as 20 mg. Further, the obtained compound 5 had the following physical properties: !H-NMR (D20, 30 ° C) 8.01 (2H, d, Fmoc), 7.80 (2H, d, Fmoc), 7.56 (4H, m, Fmoc), 5.00 (1H, d, GlcNAcl-H,), 4.95 (1H, s5 Man4'-H,), 4.84 (1H, s , Man3-H!), 4.67 (1H, d, GlcNAc2-H,), 4.56 (1H, d, G1CNAC5-H,), 4.44 (1H, d, Gal6-H,)5 4.11 (1H, dd, - 64· 1324607 (59)

Man4丨-Η2), 4.07 (1Η, dd, Man3-H2), 2.97 (1Η, bdd, Asn-β CH), 2.76 (1H, dd, NeuAc7'-H3eq), 2.62 (1H, bdd, Asn-β CH), 2.15 (12H, sx4, -Ac), 1.79 (2H, dd, NeuAc7'-H3ax); HRMS C72H1() 4N6Na043 [M + Na + ] Theory 1763.6034, found 1763.6074. Example 6 Synthesis of Compound 9 Compound 8 (40 mg, 630 μπιοί) obtained in Example 4 and 5 mg of bovine serum albumin were dissolved in 7-8 ml of HEPES buffer solution (50 mM, pH 6.0), and further Add 3 8 U of α-mannose chymase derived from Jack Bean. The solution was allowed to stand at 37 ° C for 63 hours and then lyophilized. Further HPLC (ODS column, 2.0 φ X 25 cm, solvent for the first 15 minutes for water, 16 minutes to 30 minutes for water: acetonitrile = 1 〇: 〇 to 85: 15 , 31 minutes to 45 minutes for water: The acetonitrile = 85: 15 to 80: 20 gradient, flow rate of 3 ml / min) was purified to give the desired compound 9 as 30 mg. Further, the obtained compound 9 has the following physical properties. 'H-NMR (D20, 30°C) 8.01 (2H, d, Fmoc), 7.80 (2H, d, Fmoc), 7.56 (4H, m, Fmoc), 5.23 (1H, s, Man4-Hi), 5.08 (1H, d5 GlcNAcl-H,), 4.53 (1H, d, Gal6-Hi), 4.32 (1H, dd, Man3-H2), 4.28 (1H, dd, Man4-H2)5 2.8 1 (1H, bdd, Asn-β CH), 2.76 (1H, dd, NeuAc7-H3eq), 2.59 (1H, bdd, Asn-β CH), 2.13 (12H, Sx4, -Ac), 1.80 (1H, dd, NeuAc7H3ax) ; HRMS C72H104N6NaO43 [M + Na + ] theoretical 1 763.6034, found 1 763.604 1. -65- 1324607 (60) 11⁄2 明明明 Example 7 Fmoc-based deprotection (synthesis of compound 33) The compound 1 obtained in Example 2 〇 (1 〇. 5 mg, 5 · 2 7 μm 〇1 Dissolved in 150 ml of 50% morpholine/N,N-dimercaptoacetate solution and allowed to react for two hours at room temperature under argon. To the solution was added 3 ml of a solution and placed in an evaporator at 35 °C. This step was repeated three times to 'removal of the reaction solvent. The residue was purified by a gel filtration chromatography column (Sephadex G-25, 2.5 φ χ 30 cm, developing solvent water, flow rate of 1.0 mL/min) to obtain a compound of 7 mg as shown in FIG. The compound 33 (yield: 76%) was again obtained based on the 1 Η-NMR spectrum analysis and the compound 33 obtained in Example 2, and the compound 3 3 was confirmed. The physical properties of the obtained compound 3 3 are shown below. ^-NMR (30°C) δ 5.12 (s, 1H, Man4-H-1), 5.07 (d, 1H, J = 9.7 Hz,

GlcNAcl-H-1), 4.92 (s, 1H, Man4'-H-1), 4.76 (s, 1H,

Man3-H-1), 4.62 (d, 1H, J = 8.0 Hz, GlcNAc2-H-1), 4.58 (d, • 2H, J = 7.8 Hz, GlcNAc5, 5'-Hl), 4.47 (d, 2H , J = 7.9 Hz,

Gal6, 6'-Hl), 4.24 (bd, 1H, Man3-H-2), 4.19 (bdd, 1H, J = 3.2 Hz, 1.4 Hz, Man4'-H-2), 4.12 (bdd, 1H, J = 3.2 Hz, 1.4 Hz, Man4-H-2), 2.93 (dd, 1H, J = 4.5 Hz, 17.0 Hz, Asn-β CH), 2.93 (dd, 1H, J-6.8 Hz, 17.0 Hz, Asn- β CH), 2.08 (s, 3H, Ac), 2.05 (s,6H,Ac χ 2 ),2 · 0 1 (s,3H, Ac) -66- 1324607 (61) Monthly Results Page 8 Compound 1 Synthesis of 4 Compound 3 (28 mg ' 21.3 μηι〇ι) and 1 mg of bovine serum albumin were dissolved in HEPES buffer solution (5 mM, ρΗ5·0, 454 (iL), and further added with neuroamine Acid enzyme (made by Viglio Cholerae '198 mU). After the solution was allowed to stand at 37 t: 20 hours, the reaction was confirmed by ΗPLC analysis. The reaction solution was packaged in HPLC (YMC). Column D-ODS-5 S-5 120Α ODS No. 202 0 1 78 ' 20 X 250 mm 'Expansion solvent is 50 mM aqueous ammonium acetate: acetonitrile = 80: 20, flow rate 4 ml / min) for purification. Further 'with ODS column (Cosmoki 75Cig-〇PN, 15 X 100 mm, initially dissolved in 50 liters of H2 '' again Desalting was carried out by 25% hexanes, and the desired compound 1 4 (17 mg, yield 70%) was obtained. The obtained compound 14 was obtained as follows. *H-NMR (30 ° C) ) δ 7.91 (d5 2H, J = 7.5 Hz, Fmoc), 7.71 (d, 2H, J = 7.5 Hz, Fmoc), 7.51 (dd, 2H, J = 7.5 Hz, Fmoc), 7.43 (dd, 2H, J = 7.5 Hz, Fmoc), 5.12 (s, 1H, Man4-H-1), 4.99 (d, 1H, J = 9.5 Hz, GlcNAcl-H-1), 4.92 (s, 1H, Man4'-H-1 ), 4.76 (s, 1H,

Man3-H-1), 4.58 (d, 1H, J = 8.0 Hz, GlcNAc2-H-1), 4.55 (d, 1H, J = 8.4 Hz, GlcNAc5'-H-1), 4.47 (d, 1H, J = 7.8 Hz,

Gal6'-H-1), 4.34 (t, 1H, Fmoc), 4.24 (bd, 1H, J=1.9 Hz, Man3-H-2), 4.18 (bdd, 1H, J=1.4 Hz, 3.3 Hz, Man4 -H-2), 4.11 (bdd, 1H, J=1.4 Hz, 3.5 Hz, Man4'-H-2), 2.72 (bdd, 1H, J = 3.0 Hz, 15.7 Hz, Asn-β CH), 2.52 ( Bdd, 1H, J = 8.7 -67- 1324607 (62) 13⁄4 明諕

Hz, 15.7 Hz, Asn-β CH), 2.06, 2.05, 2.04, 1.89 (each s, each 3H, Ac); HRMS CwH!, 〇N6Na045 [M + Na + ] Theoretical value 1837.6402, found 1837.6471 ° Example 9 Synthesis of Compound 1 9 Compound 7 (20 mg, 9.4 μιηοΐ) and bovine serum albumin 1.6 mg were dissolved in HEPES buffer solution (50 mM, pH 5.0, 323 pL), and further added with ceramide (Sigma-based company

'by Viblio Cholerae' 141 mU). This solution was at 37. (: After standing = 1 8 hours later) The reaction was confirmed by HPLC analysis. Then, HPLC (YMC packaging column D-ODS-5 S-5 120A ODS No. 2020178, 20 X 250mm' developing solvent was 50 mM Ammonium acetate aqueous solution: acetonitrile = 8 〇: 2 〇 'flow rate 4 ml / min) for purification. Further, 〇ds column (Cosmoki 75Ci8-OPN' 15 x 100 mm, initially dissolved in 50 ml H20, Desalting with 25% acetonitrile to obtain the desired compound i 9 (13 mg, yield 76%). The obtained compound was consistent with the standard because of the result of 1 η - NM R The structure was confirmed. Lu Example 1 0 Synthesis of Compound 1 5 Compound 4 (45 mg, 24 μπιοί) and bovine albumin 1.7 mg were dissolved together in HEPES buffer solution (50 mM, pH 5.0, 820 pL) In the 'further addition of neuraminidase (made by Sigma-Delci, Viblio Cholerae' 134 mU). The solution was allowed to stand at 37. After standing for 14 hours, the reaction was confirmed by ΗPLC analysis. Then, the reaction solution was HPLC (YMC packaging column D-ODS-5 S-5 120Α ODS No 2020 1 78 ' 20 X 250 mm, developing solvent is 50 mM ammonium acetate aqueous solution -68-1324607 (63) Description of the invention: acetonitrile = 80: 20, flow rate 4 ml / min) for purification. Further, 'the salt of the ODS column (Cosmoki 75Ci8_〇pN, 15 X 1〇〇mm 'dissolved initially in 50 ml Ηπ, then dissolved in 25% acetonitrile) to obtain the desired compound 15 (28 mg, Yield 74%) The physical properties of the obtained compound 15 are as follows: H-NMR (30 ° C) δ 7.92 (d, 2H, J = 7.5 Hz, Fmoc), 7_71 (d, 2H, J -7_5 Hz,

Fmoc), 7.51 (dd, 2H, J = 7.5 Hz, Fmoc), 7.44 (dd, 2H, J = 7.5 Hz, Fmoc), 5.10 (s, 1H, Man4-H-1), 4.99 (d, 1H, J = 9.5 Hz, GlcNAcl-H-1), 4.92 (s, 1H, Man4'-H-1), 4.76 (s, 1H,

Man3-H-1), 4.58 (ds 2H, GlcNAc2, 5'-Hl), 4.47 (d, 1H, J = 8.0 Hz, Gal6'-H-1), 4.35 (t, 1H, Fmoc), 4.24 ( Bd, 1H, J=1.9 Hz, Man3-H-2), 4.11 (bs, 1H, =Man4'-H-2), 4.07 (bs, 1H, Man4-H-2), 2.72 (bd, 1H, J=15.5 Hz, Asn-β CH), 2.52 (bdd, 1H, J = 8.7 Hz, 15.5 Hz, Asn-β CH), 2.06, 2.04, 1.89 (each s,each 3H, Ac) ; HRMS C67H97N5NaO40 [M + Na + ] theoretical value 1634.5608, found 1634.5564 Example 1 1 Synthesis of compound 7 0

Compound 15 (11 mg, 6.8 μηιοί) and 1.5 mg of bovine serum albumin were dissolved in HEPES buffer solution (50 mM, ρΗ5.0, 269 μΙ〇, and further added with stone-galactose enzyme (manufactured by Biochemical Co., Ltd.) From Jack Bean, 1 1 pL, 275 mU). After the solution was allowed to stand at 37 ° C for 14 hours, it was confirmed by HPLC analysis that the reaction was completed. The reaction solution was subjected to HPLC.

(YMC packaging column D-ODS-5 S-5 120AODSN〇. 2020178,20 X -69- 1324607 (64) ϋϋϋκΐ 2 5 0 mm, developing solvent is 50 m Μ acetic acid aqueous solution: acetonitrile = 8 Ο : 2 Purine was carried out at a flow rate of 4 ml/min. Further, desalting was carried out with a 〇DS column (Cosmoki 75C丨8-OPN, 15 x 100 mm, initially dissolved in 50 ml of H20, and then dissolved in 25% acetonitrile) to obtain the desired compound 7 〇 (6) · 3 mg, yield 64%). The physical properties of the obtained compound are shown below. ^-NMR (30 ° C)

δ 7.91 (d, 2H, J = 7.5 Hz, Fmoc), 7.70 (d, 2H, J = 7.5 Hz, Fmoc), 7.50 (dd, 2H, J = 7.5 Hz, Fmoc), 7.43 (dd, 2H, J = 7.5 Hz, Fmoc), 5.10 (s, 1H, Man4-H-1), 4.99 (d, 1H, J = 9.5 Hz, GlcNAcl-H-1), 4.9 1 (s, 1H, Man4'-H- 1), 4.76 (s, 1H,

Man3-H-1), 4.55 (d, 2H, GlcNAc2, 5'-Hl), 4.32 (t, 1H, Fmoc), 4.24 (bs, 1H, Man3-H-2), 4.10 (bs, 1H, Man4 -H-2), 4.06 (bs, 1H, J=1.3 Hz, Man4'-H-2), 2.72 (bd, 1H, J=14.0 Hz, Asn-β CH), 2.52 (bdd, 1H, J = 9.5 Hz, 14.8 Hz, Asn-β CH), 2.06, 2.05, 1.89 (each s, each 3H, Ac) ; MS (Fab) C61H88N5035 [M + H + ] Theoretical value 1450.5, found 1450 4 Example 1 2 Synthesis of Compound 20 Compound 8 (47 mg '25 μπιοί) was dissolved in HEPES buffer solution (50 mM, pH 5.0, 840 μΙ〇) with further addition of ceramide (Sigma) Made by Yadlich, Viblio Cholerae, 369 mU). The solution was allowed to stand at 37. After standing for 37 hours, the reaction was confirmed by PLC analysis. The reaction solution was freeze-dried and then HPLC. (YMC packaging column D-odsj s_5 ι2〇Α -70- 1324607 ODS No· 2020178 ' 20 X 250 mm ' Developing solvent is 50 mM ammonium acetate aqueous solution: acetonitrile = 8 0 : 2 0, flow rate 4 ml / min) Purification. Further 'to ODS official column (Cosmoki 75Ci8〇p N,15 X i〇〇nim 'is initially dissolved in 50 ml of Η2 Ο, and then dissolved in 25% acetonitrile) to obtain the desired compound 2 0 (26 mg, yield 65 %). The physical properties of the compound are as follows: 'H-NMR (30 ° C ) δ 7.92 (d, 2H, J = 7.5 Hz, Fmoc), 7.71 (d, 2H, J-7.5 Hz,

Fmoc), 7.51 (dd, 2H, J = 7.5 Hz, Fmoc), 7.43 (dd, 2H, J = 7.5

Hz, Fmoc), 5.12 (s, 1H, Man4-H-1), 4.99 (d, 1H, J = 9.4 Hz, GlcNAcl-H-1), 4.91 (s, 1H, Man4'-H-1), 4.77 (s, 1H,

Man3-H-1), 4.57 (bd, 2H, GlcNAc2, 5'-Hl), 4.46 (d, 1H, J = 7.5 Hz, Gal6'-H-1), 4.34 (t, 3H, Fmoc), 4.24 (bs, 1H, Man4'-H-2), 4.19 (bs, 1H, Man4-H-2), 2.72 (bd, 1HS

J = 15.5 Hz, Asn-β CH), 2.52 (bdd, 1H, J = 9.2 Hz, 15.5 Hz, Asn-β CH), 2.06, 2.05, 1.89 (each s, each 3H, Ac) ; HRMS C67H97N5Na04. [M + Na + ] theoretical 1 634.5608 , found 1634.5644 Example 1 3 Synthesis of Compound 7 1

Compound 20 (12 mg, 7.4 μιηοΐ) and bovine serum albumin 1.0 mg were dissolved in HEPES buffer solution (50 mM, ρΗ5.0, 330 μΙ〇, and further added to CAM-galactosidase (Biochemical Industry Co., Ltd.) The system was made up of Jack Bean, 12 μί, 297 mU. The solution was taken at 37. (: After standing for 46 hours, the reaction was confirmed by HPLC analysis. The reaction solution was HPLC-71 · 1324607 (66) ** 1 &quot; 1 ... (YMC packaging column D-ODS-5 S-5 120AODS No. 2020178, 20 χ 250 mm, developing solvent 50 mM ammonium acetate aqueous solution: acetonitrile = 80: 20 'flow rate 4 ml / Purification. Further, desalting was carried out with an ODS column (Cosmoki 75C18-OPN, 15 x 100 mm, initially dissolved in 50 ml of H 2 ,, and then dissolved in 25% acetonitrile) to obtain the desired compound 7 1 (6.6 mg, yield 61%) The physical properties of the obtained compound are as follows: *Η-ΝΜΚ (30 ° C) • δ 7.90 (d, 2H, J = 7.5 Hz, Fmoc), 7.70 (d , 2H, J = 7.5 Hz, Fmoc), 7.49 (dd, 2H, J = 7.5 Hz, Fmoc), 7.42 (dd, 2H, J = 7.5 Hz, Fmoc), 5.11 (s, 1H, Man4-H-1 ), 4 .99 (d, 1H, J = 9.4 Hz, GlcNAcl-1 H-1), 4.91 (s,1 Η, Man4'-H- 1), 4.7 6 (s, 1 H, Man3-H-1), 4.55 (d, 2H, GlcNAc2, 5-Hl), 4.31 (b, 1H, Fmoc), 4.24 (bs, 1H, Man3-H-2), 4.18 (bs, 1H, Man4-H-2), 3.97 ( Dd, 1H, J=1.8 Hz, 3.3 Hz, Man4'-H-2), 2.72 (bd, 1H, J=15.5 Hz, Asn-β CH), 2.52 (bdd, 1H, J = 8.0 Hz, 15.5 Hz ♦ Asn-β CH), 2.06, 2.05, 1.88 (each s, each 3H, Ac) ; MS (Fab) C61H88N5035 [M + H + ] Theoretical value 1 450.5, found 1 450.3 Example 1 4 Compound 1 6 Synthesis Compound 5 (32 mg, 18·4 μηιοί) and bovine serum albumin 2.5 mg were dissolved in HEPES buffer solution (50 mM, pH 5.0, 713 μ! ,, and further added with ceramide) (Sigma) Made by Delich, by Viblio Cholerae ' 134 mU). After the solution was allowed to stand at 37 ° C for 17 hours, it was confirmed by HPLC analysis that the reaction was completed. The reaction solution-72-1324607 (67) was further purified by HPLC (YMC packaging column D-ODS-5 S-5 120A ODS No. 2020178' 20 x 250 mm, developing solvent 50 mM ammonium acetate aqueous solution: acetonitrile = Purification was carried out at 8 0 : 2 0 at a flow rate of 4 ml/min. Further, desalting was carried out with an ODS column (Cosmoki 75C丨8-OPN, 15 X 100 mm, initially dissolved in 50 ml of H20, and then dissolved in 25 % acetonitrile) to obtain the desired compound 16 ( 1 3 mg, yield 52%). The physical properties of the obtained compound are as follows. 'H-NMR (30°C) δ 7.92 (d, 2H, J = 7.5 Hz, Fmoc), 7.71 (d, 2H, J = 7.5 Hz, Fmoc), 7.51 (dd, 2H, J = 7.5 Hz, Fmoc ), 7.44 (dd, 2H, J = 7.5 Hz, Fmoc), 5.00 (d, 1H, J = 9.9 Hz, G1 cNAc 1 -H-1 ), 4.92 (s, 1H, Man4'-H-1), 4.75 (s, 1H, Man3-H-1), 4.58 (d, 2H, J = 7.5 Hz, GlcNAc2, 5'-Hl), 4.47 (d, 1H, J = 7.8 Hz,

Gal6'-H-1), 4.34 (t, 1H, Fmoc), 4.10 (bd, 1H, Man3-H-2), 4.07 (bs, 1H, Man4'-H-2), 2.72 (bdd5 1H, J =15.5 Hz, Asn-β CH), 2.52 (bdd, 1H, J = 9.2 Hz, 15.5 Hz, Asn-β CH), 2.07, 2.05, 1.89 (each s, each 3H, Ac) ; MS (Fab) C61H88N5035 [M+H+] Theory 1450.5, found 1450.3. Example 1 Synthesis of Compound 1 7

Compound 16 (9 mg ' 6.2 μιηοΐ) and bovine serum albumin ι·6 mg were dissolved in HEPES buffer solution (50 mM, pH 5.0, 613 μΙ〇, and further added with /3 -galactosidase (raw) Made by Chemical Industry Co., Ltd., 186 mU by Jack Bean. After the solution was allowed to stand at 37 ° C for 32 hours, it was confirmed by HPLC analysis that the reaction was completed. The reaction solution was packed in HPLC (YMC -73 - 1324607 (68)) Column D-ODS-5 S-5 120A ODS No. 2020178, 20 χ 250 mm, developing solvent 50 mM aqueous ammonium acetate: acetonitrile = 80: 2 Torr, flow rate 4 ml / min) for further purification. Desalting with an OD S column (Cosmoki 75C18-OPN, 15 χ 100 mm, initially dissolved in 50 ml of H 2 ,, and then dissolved in 25% acetonitrile) to obtain the desired compound i 7 (5.4 mg, produced) The rate is 68%. The physical properties of the obtained compound are not shown. 】H-NMR (30〇C)

δ 7.8 9 ( d,2 Η, J = 7 · 5 Η z, F moc ) , 7.6 8 ( d, 2 Η, J = 7 · 5 Η z, F moc), 7 · 4 9 (dd, 2 H , J = 7 · 5 H z, F moc), 7 · 4 2 (dd, 2 H, J = 7.5 Hz, Fmoc), 4.99 (d, 1H, J = 9.7 Hz, GlcNAcl-H-1), 4.91 (s, 1H, Man4'-H-1), 4.55 (d, 1H, J = 8.1 Hz, GlcNAc2, 5'-Hl), 4.09, 4.07 (s, 1H, Man4'-H-2, Man3-H -2), 2.72 (bd, 1H, J=15.5 Hz, Asn-β CH), 2.56 (bdd, 1H, J = 8.1 Hz, 15.5 Hz, Asn-β CH), 2.07, 2.05, 1.89 (each s, Each 3H, Ac) ; MS (Fab) calcd for C55H77N5Na03. [M + Na + ] Theoretical value 13 10.5, found 1310.2 Example 1 Synthesis of Compound 1 8 Compound 17 (3.4 mg, 2.6 μιηοΐ) and bovine serum albumin 1.1 mg were dissolved together in HEPES buffer solution (50 mM) Further, Ν-acetamido-cold-D-glucosamine enzyme (manufactured by Sigma Delich, manufactured by Jack Bean, 144 mU) was further added to pH 5.0, 257 μΙ&gt;). After the solution was allowed to stand at 37 ° C for 24 hours, it was confirmed by HPLC analysis that the reaction was completed. The reaction solution was subjected to HPLC (YMC packaging column D-ODS-5 S, 5 120A ODS No. -74-1324607 (69). The invention page was 2020 1 78, 20 X 250 mm, and the developing solvent was 5 mM ammonium acetate. The aqueous solution was washed with acetonitrile = 80:20 at a flow rate of 4 ml/min. Further, the ODS column (Cosmoki 75C18-OPN, I5 x 100 mm 'is initially dissolved in 50 ml Η 2 ,, and then eluted with 25% acetonitrile) to obtain the desired compound 1 8 ( 2 · 1 mg, yield 7 5 %). The physical properties of the obtained compound are shown below. ^-NMR (30°C) δ 7.91 (d, 2H, J = 7.5 Hz, Fmoc), 7.71 (d, 2H, J = 7.5 Hz, Fmoc), 7.51 (dd, 2H, J = 7.5 Hz, 7.5 Hz , Fmoc), 7.43 (dd, 2H, J = 7.5 Hz, 7.5 Hz, Fmoc), 5.00 (d5 1H, J = 9.7 Hz, GlcNAc 1-H-1), 4.91 (d, 1HS J=1.6 Hz, Man4 '-H-1), 4.76 (s, 1HS Man3-H-1), 4.58 (d, 1H, J = 7.8 Hz, GlcNAc2-H-1), 4.34 (t, 1H, Fmoc), 4.07 (d, 1H, J = 2.7 Hz, Man4'-H-2), 3.97 (dd, 1H, J=1.6 Hz, 3.7 Hz, Man3-H-2), 2.72 (bdd, 1H, J = 3.2 Hz, 15.1 Hz, Asn-β CH), 2.52 (bdd, 1H, J = 8.9 Hz, 15.1 Hz, Asn-β CH), 2.07, 1.89 (each s, each 3H, Ac) ; MS (Fab) C47H65N4025 [M + Na + ] Theoretical value 1085.4, found 1 085.3 Example 1 7 Synthesis of compound 2 1 Compound 9 (28 mg, 16 μιηοΐ) was dissolved in HEPES buffer solution together with bovine serum albumin ι 7 mg (50 mM, ρΗ 5 〇, 624 In the μΐ^, a further addition of a neuraminidase (made by Vig丨io Cholerae' Η7 mU) was carried out. The solution was allowed to stand at 37°C for 17 hours, and the reaction was confirmed by HPLC analysis. End. Then, will react Solution to HPLC (YMC packed column D-0Ds_5 s_5 12QA 〇DS Nq &gt; -75 · 1324607

(70) [MSH 2020178, 20 x 250 mm, developing solvent: 50 mM aqueous solution of ammonium acetate: B gambling = 80:20, flow rate 4 ml/min) was purified. Further, desalting was carried out with an ODS column (Cosmoki 75CI8-OPN, 15 X 100 mm, initially dissolved in 50 ml Η 2 ,, and then dissolved in 25 % acetonitrile) to obtain the desired compound 2 1 ( 1 4 · 6 mg, yield 68%). The physical properties of the obtained compound are shown below. *H-NMR (30°C) δ 7.92 (d, 2H, J = 7.5 Hz, Fmoc), 7.71 (d, 2H, J = 7.5 Hz, • Fmoc), 7.50 (dd, 2H, J = 7.5 Hz, Fmoc), 7.43 (dd, 2H, J = 7.5 Hz, Fmoc), 5.12 (s, 1H, Man4-H-1), 4.99 (d, 1H, J = 9.5 Hz, GlcNAcl-lH-1), 4.77 ( s, 1H, Man 3-H -1 ), 4 · 5 7 (d, 2 H, J = 7.2 Hz, GlcNAc2-Hl), 4.46 (d, 1H, J = 7.8 Hz,

Gal6-H-1), 4.34 (t, 1H, Fmoc), 4.22 (bd, 1H, J = 2.7 Hz, Man3-H-2)s 4.19 (b, 1H, Man4-H-2), 2.72 (bdd , 1H, J=15.5 Hz, Asn-β CH), 2.52 (bdd5 1H, J = 9.8 Hz, 15.5 Hz, Asn-β CH), 2.05 (s, 6H, Acx2), 1.89 (s, 3H, Ac) MS (Fab) ® C61H88N5035 [M + H + ] Theoretical value 1 45 0.5, found 1 450.3 Example 1 8 Synthesis of compound 2 2 Compound 21 (1'0 mg, 6.9 μιηοΐ) and bovine serum albumin 1.6 The mg was dissolved in HEPES buffer solution (50 mM, pH Η 5.0, 672 μΙ〇, and further added with yS-galactosidase (manufactured by Biochemical Industries, Ltd., from Jack Bean, 205 mU). The solution was at 37 ° C. After standing for 20 hours, the completion of the reaction was confirmed by HPLC analysis. The reaction solution was subjected to HPLC (YMC packaging column D-ODS-5 S-5 120A ODS No. 2020178, 20 X 250 • 76-1324607 (71) Mm, the developing solvent is 5 〇m Μ acetic acid recording aqueous solution: acetonitrile = 8 0 : 2 0 'flow rate 4 ml / min) for purification. Further 'with 0 D s column (Cosmoji 75Ci8-〇PN , 15 X 100 mm, initially dissolved in 50 ml of H20, then dissolved in 25% acetonitrile) Desalting to give the desired compound 2 2 (5 · 6 mg 'yield 64%). The physical properties of the obtained compound are as follows: H-NMR (30 ° C) δ 7.87 (d, 2H, J = 7.5 Hz, Fmoc), 7.67 (d, 2H, J = 7.5 Hz, Fmoc), 7.48 (dd, 2H, J = 7.5 Hz, Fmoc), 7.41 (dd, 2H, J = 7.5 Hz, Fmoc), 5.12 (s, 1H, Man4-H-1), 4.99 (d, 1H, J = 9.7 Hz, GlcNAcl-H-1), 4.76 (s5 1H, Man3-H-1), 4,55 (d, 2H , J = 8.6 Hz, GlcNAc2, 5-Hl), 4.26 (t, 1H, Fmoc), 4.22 (d, 1H, J = 2.2 Hz, Man3-H-2), 4.18 (bdd, 1H, J=1.3 Hz , 3.3 Hz, Man4-H-2), 2.72 (bd, 1H, J=15.5 Hz, Asn-β CH), 2.54 (bdd, 1H, J = 9.5 Hz, 15.5 Hz, Asn-β CH), 2.05 ( s, 6H, Acx2), 1.88 (s, 3H, Ac) » MS (Fab) C55H78N5O3Q [M + H + ] Theoretical value 1288.5, found 1288.3 Example 1 9 Compound 2 Synthesis of compound 22 (3.6 mg, 2.8 μιηοΐ) and bovine serum albumin 12 mg were dissolved in HEPES buffer solution (50 mM, ρΗ5.〇, 277 μΙ;), and further Ν-acetyl-/3-D-glucosamine enzyme ( Sigma Delich Company's 'by Jack Bean, 1 9 5 mU). After the solution was allowed to stand at 37 for 24 hours, it was confirmed by HPLC analysis that the reaction was completed. The anti-corrosion solution was developed by HPLC (YMC packaging column D-ODS-5 S-5 120A 〇DS No -77-1324607 (72) I description page 2020 178,20 χ 250 mm, developing solvent 50 mM acetic acid The aqueous ammonium solution: acetonitrile = 80:20, flow rate 4 ml/min) was purified. Further, desalting was carried out with an ODS column (Cosmoki 75C18-OPN, 15 X 100 mm, initially dissolved in 50 ml of H20 and then dissolved in 25% acetonitrile) to obtain the desired compound 23 (2.3 mg, yield 7 7%). The physical properties of the obtained compound are shown below. ^-NMR (30 ° C)

δ 7.91 (d, 2H, J = 7.5 Hz, Fmoc), 7.70 (d, 2H, J = 7.5 Hz, Fmoc), 7.50 (dd, 2H, J = 7.5 Hz, Fmoc), 7.43 (dd, 2H, J = 7.5 Hz, Fmoc), 5.11 (s, 1H, Man4-H-1), 4.99 (d, 1H, J = 9.7 Hz, GlcNAcl-H-1), 4.77 (s, 1H, Man3-H-1) , 4.57 (d, 1H, J = 6.5 Hz, GlcNAc-H-1), 4.33 (t, 1H, Fmoc), 4.22 (d, 1H, J = 3.0 Hz, Man3-H-2), 4.07 (bdd, 1H, J = 2.1 Hz, Man4-H-2), 2.72 (bdd, 1H, J=15.5 Hz, Asn-β CH), 2.52 (bdd, 1H, J = 8.9 Hz, 15.5 Hz, Asn-β CH) , 2.05, 1.89 (each s, each 3H, Ac); MS (Fab) C47H65N4025 [M + H + ] calc. 1085.4, found 1 085.3 Example 20 Synthesis of Compound 1 Compound 10 (123 mg, 62 μιηοΐ ) dissolved in HEPES buffer solution (50 mM, pH 5.0, 2.5 mL) together with bovine serum albumin 1.1 mg, and further added with /5-galactosidase (manufactured by Biochemical Industries, Inc., by Jack Bean, 24 pL, 612 mU). After the solution was allowed to stand at 37 X: for 61 hours, it was confirmed by HPLC analysis that the reaction was completed. The reaction solution was freeze-dried, and then HPLC (YMC packaging column D-ODS-5 S-5 120AODS No. 2020178, 20 X 2.5 0 mm, developing solvent 50 mM ammonium acetate aqueous solution -78-1324607 (73) [53⁄4 明癀寅: Acetonitrile = 8 0 : 2 ο, flow rate of 3.5 ml / min) for purification. Further, the ODS column (Cosmoki 75C丨8-OPN, 15 X 100 mm, initially dissolved in 50 ml Η 2 ,, and then dissolved in 25% acetonitrile) is desalted to obtain the desired compound 1 i (7 1 mg, yield 70%). The physical properties of the obtained compound are shown below. JH-NMR (30°C) δ 7.91 (d, 2H, J = 7.5 Hz, Fmoc), 7.71 (d, 2H, J = 7.5 Hz, Fmoc), 7.50 (dd, 2H, J = 7.5 Hz, Fmoc) , 7.43 (dd, 2H, J = 7.5 Hz, Fmoc), 5.11 (s, 1H, Man4-H-1), 4.99 (1H, d, J = 9.9 Hz, GlcNAcl-H-1 ), 4.91 (s, 1 Η, Man4' -H-1 ), 4 · 7 6 (s, 1 H,

Man3-H-1), 4.55 (d, 2H, J-8.6 Hz, GlcNAc2, 5-Hl), 4.34 (t, 1H, Fmoc), 4.24 (s, 1H, Man3-H-2), 4.18 (s , 1H,

Man4-H-2), 4.10 (s, 1H, Man4'-H-2), 2.72 (bd, 1H, J=15.5 Hz, Asn-β CH), 2.51 (bdd, 1H, J = 9.0 Hz, 15.5 Hz, Asn-β CH), 2.06 (s, 3H, Ac), 2.05 (s, 6H, Ac&gt;&lt;2), 1.88 (s, 3H, Ac); HRMS C69H10〇N6NaO4Q [M + Na + ] Value 1675.5873, found 1675.5841 Example 2 1 Synthesis of Compound 1 2 Compound 11 (50 mg, 30 gmol) was dissolved in HEPES buffer solution with 5 mM mg of bovine serum albumin (5 mM 'pH 5.0, 920 μ In addition, Ν-acetamido-cold_D-glucosamine enzyme (manufactured by Sigma-Delci, from Jack Bean, 2.1 U) was further added. The solution was allowed to stand at 371 for 48 hours. The reaction was confirmed by HPLC analysis. The reaction solution was developed by HPLC (YMC packaging tube D-ODS-5 S-5 120A ODS No. -79- 1324607 _ (74) 2020 1 78, 20 χ 250 mm, developing solvent Purified by 50 mM aqueous ammonium acetate solution: acetonitrile = 80:20, flow rate of 4 ml/min., and then lyophilized. The residue was taken as an ODS column (Cosmoki 75C 丨 8-OPN, 15 x 100 mm). , initially dissolved in 50 ml Η2 Ο, and then dissolved in 25% acetonitrile) Desalting gave the desired compound 12 (25 mg, yield 66%). The physical properties of the obtained compound are as follows: !H-NMR (30 °c)

δ 7.91 (d, 2H, J = 7.5 Hz, Fmoc), 7.70 (d, 2H, J = 7.5 Hz, Fmoc), 7.50 (dd, 2H, J = 7.5 Hz, Fmoc), 7.43 (dd, 2H, J = 7.5 Hz, Fmoc), 5.10 (s, 1H, Man4-H-1), 4.99 (d, 1H, J = 9.7 Hz, GlcNAcl-H-1), 4.91 (bd, 1H, J=1.6 Hz, Man4 '-H-1), 4.77 (s,1H, Man3_H-l), 4_58~4.52 (b, 1H, GlcNac2-Hl). 4.33 (t, 1H, Fmoc), 4.24 (bs, 1H, Man3-H- 2), 4.06 (dd 1H, J=1.6 Hz, 3.2 Hz, Man4-H-2), 3.97 (dd, 1H, 3=1.6 Hz, 3.5 Hz, Man4'-H-2), 2.72 (bd, 1H , J = 15.5 Hz, Asn-β CH), 2.53 (bdd, 1H, J = 9.0 Hz, 15.5 Hz, Asn-β CH), 2.05, 1.88 (each s, each 3 H, Ac) Example 2 2 Compound Synthesis of 1 3 Compound 12 (10 mg, 11 μηι〇ι) and bovine serum albumin 〇 9 mg were dissolved in HEPES buffer solution (50 mM, pH 5.0, 440 μ! ,, further added by α · The mannose enzyme (manufactured by Sigma Bean, 30 μί, 3.2 U) was used to 37. (: After standing for 21 hours), it was confirmed by HPLC analysis that the reaction was complete. Then hplC (YMC packaging column D-ODS-5 S-5 120A ODS No. 2020178, 20 χ 250 mm -80- 132 4607

(75) SiMSiM was developed by purifying the solvent to a 50 mL aqueous solution of acetic acid: acetonitrile = 8 0: 2 Torr, at a flow rate of 4 ml/min. It was desalted by a 〇DS column (Cosmoki 75C18-OPN, 15 x 100mm, initially dissolved in 50 ml of H20 and then dissolved in 25% acetonitrile) to obtain the desired compound 13 (3 mg, produced). Rate 43%). The physical properties of the obtained compound are as follows. H-NMR (30 ° C) δ 7.92 (d, 2H, J = 7.5 Hz, Fmoc), 7.71 (d, 2H, J = 7.5 Hz, Fmoc), 7.51 (dd, 2H, J = 7.5 Hz, Fmoc ), 7.43 (dd, 2H, J = 7.5 Hz, Fmoc), 4.99 (d, 1H, J = 9.5 Hz, G1 cNAc 1 -H-1), 4.76 (s, 1H, Man3-H-1), 4.57 (1H, G1 cN Ac 2 - H - 1), 4.06 (d, 1H, J = 3.2 Hz, Man3-H-2)5 2.72 (bd, 1H, J=15.5 Hz, Asn-β CH), 2.52 ( Bdd, 1H, J = 8.3 Hz, 15.5 Hz, Asn-β CH), 2.05, 1.89 (each s, each 3 H, Ac) (deprotection of the Fmoc group of the sugar chain aspartic acid derivative) Deprotection of the Fmoc group was carried out on the entire sugar chain aspartate derivative in the following order. First, for each sugar chain aspartame Fm〇c 1 μηι〇ΐ, 240 μM of N,N-dimethylformamide and 160 μM of morpholine were added and allowed to stand at room temperature with argon. The reaction is carried out under the environment. After confirming the completion of the reaction by TLC (developing solvent: 1% ammonium acetate: isopropyl alcohol = 8:5), it was cooled with ice water. After the reaction solution was added with 1 〇 amount of the dimethyl bond, the mixture was stirred for 15 minutes, and the precipitate was filtered. The resulting residue was dissolved in water at 35. Let it vaporize. The addition of 3 ml of bismuth was repeated three times for the aforementioned evaporation vaporization step. The residue is placed on the reverse phase column (Cosmoki 75C18-OPN, 15 x 100mm, the solvent is water) 1324607 _

Purification was carried out in (76) I 赛 iiH. Example 2 Synthesis of Compound 3 3 Compound 10 (10.5 mg, 5.3 μιτιοί) was reacted for 7 hours in the above-mentioned procedure to give the objective compound 33 (7 mg, yield 76%). The obtained compound was confirmed to be consistent with the standard by 1H-NMR. Example 24 Synthesis of Compound 26 Compound 3 (8.0 mg, 3.8 μηηοΐ) was reacted for 21 hours to give the objective compound 26 (6.3 mg, yield 88%). The physical properties of the compounds obtained are as follows. 'H-NMR (30 ° C) 5 5.13 (s, 1H, Man4-H-1), 5.07 (d, 1H, J = 9.9 Hz,

GlcNAcl-H-1), 4.95 (s, 1H, Man4'-H-1), 4.78 (s, 1H,

Man3-H-1), 4.62 (2H, GlcNAc2, 5'-Hl), 4.56 (d, 1H, J = 8.1 Hz, GlcNAc5-H-1), 4.52 (d, 1H, J = 7.8 Hz, Gal6' -Hl), 4.25 (bs, 1H, Man3-H-2), 4.19 (bs, 1H, Man4'-H-2), 4.12 (bs3 1H, Man4-H-2), 2.94 (dd, 1H, J = 4.5 Hz, 17.0 Hz, Φ Asn-β CH), 2.85 (dd, 1H, J = 6.8 Hz, 17.0 Hz, Asn-β CH), 2.68 (dd, 1H, J = 4.6 Hz, 12.4 Hz, Neu A c 7 ' - H - 3 eq), 2.08, 2.07, 2.06, 2.04, 2.02 (each s, each 3H, Ac), 1.72 (dd, 1H, J=12.1 Hz, 12.1 Hz, NeuAc7'-H-3 ax ; MS (Fab) C7 丨Hn8N7051 [y + H + ] Theory 1 884.7, found 1 884.5 Example 25 Synthesis of Compound 27 Compound 4 (11.0 mg, 5.8 μιηοΐ) was reacted in the above procedure. The compound of interest 2 7 (8.5 mg, yield 8 8 %) -82 - 1324607 (77) was obtained. The physical properties of the obtained compound are as follows. !H-NMR (30°c) δ 5.11 (s, 1H, Man4-H-1), 5.08 (d, 1H, J = 9.7 Hz, GlcNAcl-H-1), 4.95 (s, 1H, Man4'- H-1), 4.78 (s, 1H,

Man3-H-1), 4.62 (d, 2H, GlcNAc2, 5'-Hl), 4.45 (d, 1H, J = 7.6 Hz, Gal6'-H-1), 4.26 (bd, 1H, Man3-H- 2), 4.12 (bd, 1H, Man4'-H-2), 4.08 (bdd, 1H, J=1.6 Hz, 3.3 Hz, Man4-H-2), 2.94 (dd, 1H, J = 4.0 Hz, 17.2 Hz, Asn-β CH), 2.85 (dd, 1H, J = 7.2 Hz, 17.2 Hz, Asn-β CH), 2.68 (dd, 1H, J = 4.1 Hz, 12.1 Hz, NeuAc7'-H-3eq), 2.09, 2.07, 2.04, 2.02 (each s, each 3H, Ac), 1.72 (dd, 1H, J=12.1 Hz, 12.1 Hz,

NeuAc7'-H-3ax) ; MS (Fab) C63H104N6Na〇46 [M + Na + ] Theory 1 7 0 3 · 6, Measured 1 7 0 3 · 1 Example 2 6 Synthesis of Compound 2 8

Compound 5 (7.0 mg, 4.0 μιηοΐ) was reacted for 21 hours to give the title compound 28 (5.3 mg, yield 87%). The physical properties of the obtained compound are as follows. !H-NMR (30°c) 5 5.07 (d, 1H, J = 9.4 Hz, GlcNAcl-H-1), 4.94 (s, 1H,

Man4'-H-1), 4.76 (s, 1H, Man3-H-1), 4.61, 4.59 (each d, each 1H, GlcNAc2, 5'-H-1), 4.44 (d, 1H, J = 7.8 Hz,

Gal6'-H-1), 4.10, 4.07 (each 1H, Man4', 3-H-2), 2.93 (dd, 1H, J = 4.6 Hz, 17.5 Hz, Asn-β CH), 2.85 (dd, 1H , J = 7.0 Hz, 17.5 Hz, Asn-β CH), 2.67 (dd, 1H, J = 4.6 Hz, 12.2 Hz, -83- 1324607 _ (78) 13⁄4

NeuAc7'-H-3eq), 2.08, 2.06, 2.02, 2.01 (each s, each 3H, Ac), 1.71 (2H, dd, J=12.2 Hz, 12.2 Hz, N eu A c 7' - H - 3 ax MS (Fab) C57H94N6Na04 [M + Na + ] calcd. 1 54 1 .5, found 154 1.3 Example 2 7 Synthesis of Compound 3 Compound 7 (13.9 mg, 6.6 μιηοΐ) The reaction was carried out for 7 hours to obtain the objective compound 30 (8.0 mg, yield 46%). The physical properties of the obtained compound are as follows.丨H-NMR (30.(:) δ 5.13 (s, 1Η, Man4-H-1), 5.06 (d, 1H, J = 9.9 Hz, GlcNAcl-H-1), 4.9 1 (s, 1H, Man4'-H-1), 4.77 (s, 1H,

Man3-H-1), 4.61, 4.60 (each d, each 1H, J = 8.0 Hz, GlcNAc2, 5-Hl), 4.55 (d, 1H, J = 8.4 Hz, G1 cN A c 5 ' - H - 1 ), 4.44 (d, 1H, J = 8.0 Hz, Gal6-H-1), 4.24 (bd, 1H, Man3-H-2), 4.19 (bdd, 1H, J=1.3 Hz, 3.2 Hz, Man4'- H-2), 4.10 (bdd, 1H, J=1.4 Hz, 3.2 Hz, Man4-H-2), 2.90 (dd, 1H, • J = 4.5 Hz, 16.7 Hz, Asn-β CH), 2.80 (dd ,1H,J = 7.5 Hz, 16.7 Hz, Asn-β CH), 2.66 (dd, 1H, J = 4.6 Hz, 12.4 Hz, NeuAc7-H-3eq), 2.07, 2.06, 2.05, 2.02, 2.0 1 (each s, each 3H, Ac), 1.71 (dd, 1H, J = 12.4 Hz, 12.4 Hz, N eu A c 7 - H - 3 ax) ; MS (Fab) C71H117N7Na051 [M + Na + ] theoretical value 1 906.7, Found 1906.1 Example 2 Synthesis of Compound 3 1 Compound 8 (8.0 mg, 4.2 μηηοΐ) was subjected to the above procedure, and the compound was subjected to the above-mentioned procedure for 12 hours to obtain the objective compound 31 (6.0 mg). , yield 86%). The physical properties of the obtained compound are as follows. 'H-NMR (30°C) δ 5.12 (s, 1H, Man4-H-1), 5.06 (d, 1H, J = 9.5 Hz,

GlcNAcl-H-1), 4.91 (s, 1 H, Man4, -H-1 ), 4 · 7 7 ( s, 1H, Man3-H-1), 4.61, 4.59 (each, d, each 1H, GlcNAc2 , 5-Hl), 4.43 (d, 1H, J = 8.0 Hz, Gal6-H-1), 4.24 (bd, 1H,

Man3-H-2), 4.18 (bdd, 1H, Man4'-H-2), 2.91 (bd, 1H, J=17.0 Hz, Asn-β CH), 2.81 (dd, 1H, J = 6.5 Hz, 17.0 Hz, Asn-β CH), 2.66 (dd, 1H, J = 4.6 Hz, 12.6 Hz, NeuAc7-H-3eq), 2.06, 2.06, 2.02, 2.00 (each s, each 3H, Ac), 1.70 (dd, 1H, J-12.6 Hz, 12.6 Hz, NeuAc7-H-3 ax) ; MS (Fab) C63H 丨. 4N6Na046 [M + Na + ] Theoretical value 1 703.6, found 1 703.0 Example 2 9 Synthesis of compound 3 2 Compound 9 (7.7 mg, 4.4 μηηοΐ) was reacted in the above procedure for 23 hours to obtain the compound of interest. 3 2 (5 · 2 mg, yield 7 8 %). The physical properties of the obtained compound are as follows. !H-NMR (30°C) δ 5.14 (s, 1H, Man4-H-1), 5.07 (d, 1H, J = 9.4 Hz,

GlcNAcl-H-1), 4.78 (s, 1H, Man3-H-1), 4.61, 4.60 (each, d, each 1 H, G1 cN A c 2 , 5 - H -1), 4 _ 4 4 ( d, 1 H, J= 8.0 H z,

Gal6-H-1), 4.23 (d, 1H, J = 3.0 Hz, Man3-H-2), 4.19 (bdd, 1H, J=1.3 Hz, 2.9 Hz, Man4-H-2), 2.92 (dd, 1H, J = 4.1 Hz, 17.2 Hz, Asn-β CH), 2.83 (dd, 1H, J = 7.5 Hz, 12.7 Hz, • 85 - 1324607 (80) 13⁄43⁄43⁄43⁄4

Asn-β CH), 2.67 (dd, 1H, J = 4.6 Hz, 12.7 Hz, NeuAc7 -H-3eq), 2.06 (s, 6H, Acx2), 2.03, 2.01 (each s, each 3H, Ac), 1.71 (dd, 1H, J = 12.7 Hz, 12.7 Hz, Neu Ac7-H-3 ax); MS (Fab) C57H94N6Na04 丨 [M + Na + ] Theory 1 54 1 .5, found 1541.2 Example 3 0 Compound Synthesis of 3 7 The compound 14 (9.1 mg, 5.0 μιηοΐ) was reacted for 13 hours to give the objective compound 37 (6.5 mg, yield 77%) #. The physical properties of the obtained compound are as follows. JH-NMR (30°C) δ 5.11 (s, 1H, Man4-H-1), 5.06 (d, 1H, J = 9.5 Hz,

GlcNAcl-H-1), 4.92 (s,1 H,Man4,-H-1 ), 4 · 7 5 (s,1H,

Man3-H-1), 4.6 1, 4.57, 4.5 5 (each d, each 1H, J = 7.5 Hz, GlcNAc2, 5, 5'-Hl), 4.46 (d, 1H, J = 7.3 Hz, Gal6'- H-1), 4.23 (bs, 1H, Man3-H-2), 4.18 (bs, 1H, Man4'-H-2), 4.10 (bs, 1H, Man4-H-2), 2.87 (dd, 1H , J = 4.8 Hz, 17.0 Hz, Asn-β CH), 2.76 (dd, 1H, J = 7.2 Hz, 17.0 Hz, Asn-β CH), 2.07 (s, 3H, Ac), 2.04 (s, 6H , Acx2), 2.00 (s, 3H, Ac); MS (Fab) C6〇Hi〇0N6Na043 [M + Na + ] Theory 161 5.6, found 1615.0 Example 3 1 Synthesis of Compound 4 2 Compound 19 (9.8 </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> <RTIgt; The physical properties of the obtained compound are as follows. •86- 1324607 (81)

SSIS !H-NMR (30 ° C ) δ 5.11 (s, 1H, Man4-H-1), 5.06 (d, 1H, J = 9.5 Hz,

GlcNAcl-H-1), 4.91 (s,1H, Man4,-H-1 ), 4.76 (s,1H,

Man3-H-1), 4.60, 4.57, 4.5 5 (each d,each 1H, GlcNAc2,5, 5'-Hl), 4.46 (d, 1H, J = 7.8 Hz, Gal6-H-1), 4.28 ( s, 1H, Man3-H-2), 4.18 (s, 1H, Man4'-H-2), 4.10 (s, 1H,

Man4-H-2), 2.88 (dd, 1H, J = 4.0 Hz, 16.6 Hz, Asn-β CH), 2.77 (dd, 1H, J = 7.5 Hz, 16.6 Hz, Asn-β CH), 2.07 (s , 3H, Ac), 2.04 (s, 6H, Acx2), 2.00 (s, 3H, Ac) ; MS (Fab) C60H1()1N6O43 [M + H + ] Theoretical value 1 5 93.6, measured value 1 5 93.8 Example 3 2 Synthesis of Compound 3 8 Compound 15 (5.1 mg, 3.2 μπιοί) was reacted in the above-mentioned procedure for 1 hour to give the objective compound 38 (4.0 mg, yield: 91%). The physical properties of the obtained compound are as follows. 'H-NMR (30°C) δ 5.10 (s, 1H, Man4-H-1), 5.07 (d, 1H, J = 9.4 Hz,

GlcNAcl-H-1), 4.92 (s, 1H, Man4'-H-1), 4.76 (s, 1H,

Man3-H-1), 4.61, 4.57 (each d, each 1H, J = 7.8 Hz,

GlcNAc2, 5'-Hl), 4.47 (d5 1H, J-7.8 Hz, Gal6' Hl), 4.24 (d, 1H, J = 2.3 Hz, Man3-H-2), 4.10, 4.06 (each bd, each 1H , Man4'5 4-H-2), 2.90 (dd5 1H, 3 = 4.2 Hz, 16.8 Hz, Asn-β CH), 2.81 (dd, 1H, J = 7.3 Hz, 16.8 Hz, Asn-β CH), 2.07, 2.04, 2.01 (each s,each 3 H, Ac) ; MS (Fab) C 52Η88Ν5038 [M + H + ] Theory 1 390.5, found 1 390.1 -87 - 1324607 (82) 丨Inventive Example 33 Compound Synthesis of 72 Compound 70 (4.0 mg, 2.8 μηιοί) was reacted in the above-mentioned procedure for 13 hours to give the desired compound 7 2 (2·9 mg, yield: 85 %). The physical properties of the obtained compound are as follows. ^-NMR (30°C) δ 5.09 (s, 1H, Man4-H-1), 5.06 (d, 1H, J = 9.8 Hz, GlcNAcl -Hl), 4.91 (s, 1H, Man4'-Hl)s 4.76 (s, 1H, Man3-H-1), 4.61, 4.54 (each d, each 1H, GlcNAc2, 5-Hl), 4.24 (s, 1H, • Man3-H-2), 4.10, 4.06 (each bs , each 1H, Man4, 4'-H-2), 2.87 (dd, 1H, J-17.2 Hz, Asn-β CH), 2.76 (dd, 1H, J = 6.5 Hz, 17.2 Hz, Asn-β CH) , MS7 (Fab) C46H78N5O33 [M + H + ], , 3.3 μηιοί) In the above procedure, the reaction was carried out for 1 hour to obtain the objective compound 43 (4.1 mg, yield 87%) Φ. The physical properties of the obtained compound are as follows. ^-NMR (30°C) δ 5.11 (s, 1H, Man4-H-1), 5.07 (d, 1H, J = 9,5 Hz,

GlcNAcl-H-1), 4.91 (s, 1H, Man4'-H-1), 4.77 (s, 1H,

Man3-H-1), 4.61, 4.57 (each d, each 1H, GlcNAc2, 5-Hl), 4.46 (d, 1H, Gal6-H-1), 4.24 (s, 1H, Man3-H-2), 4.18 (bs, 1H, Man4-H-2), 2.90 (dd, 1H, J = 4.0 Hz, 17.0 Hz, Asn-β CH), 2.80 (dd, 1H, J = 7.3 Hz, 17.0 Hz, Asn-β CH), 2.07, -88 - 1324607 (83) 2.04, 2.01 (each s, each 3H, Ac); MS (Fab) C52H88N5038 [M + H + ] Theory 1390.5, found 1390.2 Example 3 5 Compound 7 3 The compound 71 (4.0 mg, 2.8 μπιοί) was subjected to the above-mentioned procedure, and the reaction was carried out for 13 hours to obtain the objective compound 7 3 (2·9 mg, yield: 85 %). The physical properties of the obtained compound are as follows. 'H-NMR (30°C) δ 5.11 (s, 1H, Man4-H-1), 5.06 (d, 1H, J = 9.9 Hz,

GlcNAcl-H-1), 4.91 (s, 1H, Man4'-H-1), 4.77 (s, 1H, Man3 -Hl), 4.60, 4.54 (each d, each 1H, J = 7.9 Hz, GlcNAc2, 5 -Hl), 4.24 (s, 1H, Man3-H-2), 4.18 (dd, 1H, J=1.6 Hz, 1.6 Hz, Man4-H-2), 3.96 (1H, dd, J=1.6 Hz, 1.6 Hz,

Man4-H-2), 2.88 (dd, 1H, J-4.3 Hz, 16.8 Hz, Asn-β CH), 2.77 (dd, 1H, J = 7.2 Hz, 16.8 Hz, Asn-β CH), 2.06, 2.04 2.00 (each s, each 3H, Ac) ; MS (Fab) C46H78N5033 [M + H + ] Theoretical value 1 2 2 8 · 5, found 1 2 2 8.3 Example 3 6 Synthesis of compound 3 9 Compound 16 ( 2.2 mg, 1.5 μηιοί) In the above procedure, the reaction was carried out for 7 hours to give the desired compound 3 9 (1.6 mg, yield: 84%). The physical properties of the obtained compound are as follows. *H-NMR (30eC) δ 5.07 (s, 1 Η, J = 9.7 Hz, GlcN Ac 1-Η-1), 4 · 92 (s, 1 H, Man4'-H-1), 4.75 (s, 1H, Man3-H- 1 ), 4,62, 4.5 8 (each d, each 1H, GlcNAc2, 5-Hl), 4.09, 4.08 (each s, each 1H, -89- 1324607 _ (84) M j

Man3, 4'-H-2), 2.91 (dd, 1H, J = 4.1 Hz, 16.9 Hz, Asn-β CH), 2.81 (dd, 1H, J = 6.8 Hz, 16.9 Hz, Asn-β CH), 2.08, 2.04, 2.01 (each s, each 3H, Ac); MS (Fab) C46H77N5Na033 [M + Na + ] Theory 1 250.4, found 1 25 0.3 Example 3 7 Synthesis of compound 40 Compound 17 (1.5 In the above procedure, the reaction was carried out for 14 hours to obtain the objective compound 40 (1·1 mg, yield: 9%). The physical properties of the obtained compound are as follows. • ]H-NMR (30°C) δ 5.07 (d, 1H, J = 9.5 Hz, G1 cN A c 1 - H -1), 4.91 (s, 1 H,

Man4'-H-1), 4.76 (s, 1H, Man3-H- 1), 4.62, 4.55 (each d, each 1H, GlcNAc2, 5-Hl), 4.10, 4.07 (each s, each, 1H, Man4's 3-H-2), 2.89 (dd, 1H, J = 3.7 Hz, 17.0 Hz, Asn-β CH), 2.79 (dd, 1H, J = 7.0 Hz, 17.0 Hz, Asn-β CH), 2.07, 2.05 , 2.01 (each, s, each 3H, Ac); MS (Fab) C4〇H67N5Na028 [M + Na + ] Theory 1088.4, found 1 088_2 _ Example 3 8 Synthesis of Compound 4 1 Compound 18 (1.3 mg , 1.2 μηιοί) In the above procedure, the reaction was carried out for 14 hours to obtain the objective compound 4 1 (0·8 mg, yield 80%). The physical properties of the obtained compound are as follows. 'H-NMR (30 ° C) δ 5.07 (d, 1H, J = 9.5 Hz, GlcNAcl-H-1), 4.91 (s, 1H,

Man4'-H-1), 4.76 (s,1H,Man3-H-1), 4.62 (d, 1H, J = 7.8 Hz, GlcNAc2-H-1), 4.08 (d, 1H, J = 2.9 Hz, Man3-H-2), *90- 1324607 i Invention Description (85)

2.92 (dd5 1H, J = 3.9 Hz, 17.3 Hz, Asn-β CH), 2.83 (dd, 1H, J = 7.0 Hz, 17.3 Hz, Asn-β CH), 2.07, 2.01 (each s, each 3H

Ac); MS (Fab) C32H55N4 〇27 [M + H + ] calc. 863.3, found 863.2 Example 3 9 Synthesis of Compound 4 4 Compound 21 (2.3 mg, 1.6 μιηοΐ) was reacted in the above step ' The objective compound 4 4 (1.6 mg 'yield 84%) was obtained in an hour. The physical properties of the obtained compound are as follows.

H-NMR (30 ° C) δ 5.11 (s, 1H, Man4-H-1), 5.06 (d, 1H, J = 9.8 Hz,

GlcNAcl-H-1), 4.77 (s, 1H, Man3-H-1), 4.61, 4.57 (each d, each 1H, GlcNAc2, 5-H-l), 4.46 (d, 1H, J = 7.8 Hz,

Gal-H-1), 4.22, 4.18 (each bs, each 1H, Man3, 4-H-2), 2.91 (dd, 1H, J = 4.1 Hz, 17.3 Hz, Asn-β CH), 2.82 (dd, 1H, J = 7.0 Hz, 17.3 Hz, Asn-β CH), 2.05, 2.04, 2.0 1 (each s,

Each 3H, Ac) ; MS (Fab) C46H78N5033 [M + H + ] </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> <RTIgt; The reaction was carried out for 14 hours to obtain the objective compound 45 (1.1 mg, yield: 85%). The physical properties of the obtained compound are as follows. 'H-NMR (30°C) δ 5.12 (s, 1H, Man4-H-1), 5.07 (d, 1H, J = 9.7 Hz, GlcNAcl -Hl), 4.77 (s, 1H, Man3-H-1 ), 4.61, 4.54 (each d, each -91 - 1324607 _ (86) Performance page: 1H, GlcNAc2, 5-Hl), 4.22 (d, 1H, J = 2.5 Hz, Man3-H-2), 4.18 ( Dd, 1H, J=1.4 Hz, 3.0 Hz, Man4'-H-2), 2.89 (dd, 1H, J = 4.3 Hz, 16.9 Hz, Asn-β CH), 2.78 (dd, 1H, J = 7.5 Hz , 16.9 Hz, Asn-β CH), 2.06, 2.05, 2.0 1 (each s,each 3H, Ac) ;MS (Fab) C40H67N5N horse 028 [M + Na + ] theoretical value 1 088.4, measured 1 0 8 8.3 . Example 4 1 Synthesis of Compound 4 6 Compound 23 (1.6 mg, 1.5 μιηοΐ) was subjected to the above-mentioned procedure to carry out a reaction for 14 hours to obtain the objective compound 46 (1.1 mg, 6.4 μπιοί, yield 8 5 . The physical properties of the obtained compound are shown below. *H-NMR (30 ° C) δ 5.10 (s, 1H, Man4-H-1), 5.06 (d, 1H, J = 9.5 Hz, GlcNAcl -Hl), 4.77 (s, 1H, Man3-H-1), 4.61 (d, 1H, J = 7.3 Hz, GlcNAc2-Hl), 4.22 (d, 1H, J-2.4 Hz, Man3-H-2), 4.07 (dd , 1H, J=1.6 Hz, 3.0 Hz, Man4'-H-2), 2.90 (dd, 1H, J = 4.3 Hz, 17.0 Hz, Asn-β CH), 2.80 (dd, 1H, J = 7.0 Hz, • 17.2 Hz, Asn-β CH), 2.05, 2.0 1 (each s, each 3H, Ac); MS (Fab) C32H55N4023 [M + H + ] Theory 863.3, found 863.3 Example 4 2 Compound 3 4 Synthesis Compound 11 (12.4 mg, 7.5 μπιοί) was reacted for 1 hour in the above-mentioned procedure to give the objective compound 34 (9.2 mg, yield 86%). The physical properties of the obtained compound are as follows. H-NMR (30°c) δ 5.11 (s, 1H, Man4-H-1), 5.07 (d, 1H, J=10.0 Hz, -92- 1324607 (87)

GlcNAcl-H-1), 4.91 (s, 1H, Man4'-H-1), 4.77 (s, 1H,

Man3-H-1), 4.61 (d, 1H, J = 6.8 Hz, GlcNAc2-H-1), 4.55 (d, 2H, GlcNAc5, 5'-Hl), 4.24 (bs, 1 H, Man 3 - H · 2), 4.1 8 (bs, 1H, Man4, -H-2), 4.10 (bs, 1H, Man4-H-2), 2.80 (dd, 1H, J = 3.8 Hz, 15.6 Hz, Asn-β CH ), 2.63 (dd, 1H, J = 8.2 Hz, 15.6 Hz, Asn-β CH), 2.07 (s, 3H, Ac), 2.05 (s, 6H, Acx2), 2.01 (s, 3H, Ac) ; MS (Fab) C54H90N6NaO38 [M + Na + ] </ </RTI> </RTI> </RTI> </RTI> </RTI> </RTI> </RTI> </RTI> </RTI> </RTI> </RTI> 5 (7.0 mg, yield 81%). The physical properties of the obtained compound are as follows. !H-NMR (30°C) δ 5.10 (s, 1H, Man4-H-1), 5.07 (d, 1H, J = 9.7 Hz,

GlcNAcl-H-1), 4.91 (s, 1 H, Man4, -H-1 ), 4 · 7 8 ( s, 1H,

Man3-H-1), 4.61 (d, 1H, J = 8.0 Hz, GlcNAc2-H-1), 4.25 (bs, 1H, Man3-H-2), 4.06 (bs, 1H, Man4, -H-2 ), 3.97 (bs, 1H, Man4-H-2), 2.79 (dd, 1H, J = 5.0 Hz, 17.0 Hz, Asn-β CH), 2.61 (dd, 1H, J = 7.3 Hz, 17.0 Hz, Asn -β CH), 2.07, 2.01 (each s,each 3H,Ac) ; MS (Fab) C38H65N4028 [M + H + ] Theoretical value 1 0 2 5.4, found 1 0 2 5.2 Example 44 Compound 76, 77 Synthesis and Separation Compounds 2 and 6 (5.0 mg '2.2 μηιοί) were dissolved in 220 pL of water t, and a 22 mM aqueous solution of cesium carbonate was added to 100 pL to adjust the pH to 7.0. -93- 1324607 (88) 13⁄43⁄43⁄43⁄4 Cool the solution; dry to the east, add N,N-dimethyl fluorene 430 μί' to the solid after drying and add 66 μηιοί thiol bromide/N,N• Diterpenoids solution 20 pL. This solution was mixed under an argon atmosphere. After 48 hours, after confirming disappearance of the starting material by TLC (developing solvent: 1NI NH4OAc: isopropyl alcohol = 2:1), 4 - 4 ml of diethyl ether was added to precipitate the compound. The precipitated sugar chain was filtered, and the residual sugar chain was dissolved in water and lyophilized. The lyophilized residue was separated and purified by HPLC (YMC packaging column D-ODS-5 S-5 120A ODS No. 2020 1 7 8, 20 X 250 mm, developing solvent: 50 m Μ acetic acid aqueous solution: When acetonitrile = 7 8 : 2 2 'flow rate: 4.0 ml/min.), when purification was carried out, compound 77' was dissolved after 8 8 minutes and 91 minutes after dissolution of compound 76. The respective ODS column (Cosmoki 75Ci8-〇PN, 15 X 100 mm 'dissolved initially in 50 ml of H2 ,, and then dissolved in 25% acetonitrile) was desalted to obtain the purpose 1 · 6 mg of compound 7 6, and 1 · 8 mg of compound 7 7. The physical properties of the obtained compound are as follows. Data for Compound 76 ♦ JH-NMR (30°C) δ 7.92 (d, 2H, J = 7.5 Hz, Fmoc), 7.71 (d, 2H, J = 7.5 Hz, Fmoc), 7.5 3 -7.40 (m, 9H , Fmoc, -CH2-Ph_), 5.38 (d5 1H, J=12.1 Hz, -CH"Ph). 5.3 1 (d, 1H, J=12.1 Hz, -CH,-Ph). 5.12 (s, 1H, Man4-H-1), 4.99 (d, 1H, J = 9.5 Hz, GlcNAcl -Hl), 4.92 (s, 1H, Μαη4·-Η-1), 4.76 (s, 1H, Man3-H-1), 4.58 (m, 3H, GlcNAc2, 5, 5'-Hl), 4.47 (d, 1H, J = 7.9 Hz, Gal6'-H-1), 4.33 (d, 1H, J = 7.9 Hz, Gal6-H- 1), 4.24 (bss •94- 1324607 ~ 'r..r ·. Τ7.Ϊ-/-.Τ; »..-.11 ,·ι&gt;τ,-τ.&quot;ίΛ.ι-*ϊ .:.1 (89) I issued a statement on page 1HS Man3-H-2), 4.19 (bs, 1H, Man4'-H-2), 4.11 (bs, 1H, Man4-H-2), 2.72 (bd, 1H, Asn-β CH), 2.68 (dd, 1H, J = 4.6 Hz, 12.7 Hz, NeuAc7-H-3eq), 2.52 (dd, 1H, J-8.7 Hz, 15.0 Hz, Asn-β CH ), 2.0 7,2.04, 2.03 , 2 · 02, 1.89 (each s, each 3H, Ac), 1.84 (dd, 1H, J=12.7 Hz, 12.7 Hz, NeuAc7-H3ax) ;MS (Fab) C99H143N17Na038 [M + H + ] Theoretical value 23 80.8, found 23 80.0.

The data of the compound 7 7 ^-NMR (30 ° C) δ 7.9 1 (d, 2H, J = 7.5 Hz, Fmoc), 7.7 1 (d, 2H, J = 7.5 Hz, Fmoc), 7.53-7.41 (m, 9H, Fmoc, -CH,-Ph), 5.37 (d, 1H, J=12.1 Hz, -CH.-Ph). 5.31 (d, 1H, J=12.1 Hz, -CH?-Ph). 5.12 (s , 1H, Man4-H-1), 4.99 (d, 1H, J = 9.8 Hz,

GlcNAcl-H-1), 4.93 (s, 1H, Man4'-H-1), 4.76 (s, 1H,

Man3-H-1), 4.58 (m, 3H, GlcNAc2, 5, 5'-Hl), 4.46 (1H, d, J = 7.8 Hz, GaI6-Hl), 4.33 (d, 1H, J-7.8 Hz, Gal6'-H-1), 4.24 (bs, 1H, Man3-H-2), 4.19 (bs, 1H, Man4'-H-2), 4.1 1 (bs, 1H, Man4-H-2), 2.72 (bd, 1H, Asn-β CH), 2.68 (dd, 1H, J = 4.8 Hz, 13.0 Hz, NeuAc7-H-3eq), 2.52 (bdd, 1H, J = 9.7 Hz, 14.1 Hz, Asn-β CH ), 2.06, 2.05, 2.04, 2.02, 2.02, 1.89 (each s5 each 3H, Ac), 1.84 (dd, 1H, J=13.0 Hz, 13.0 Hz, NeuAc7-H-3ax) ; MS (Fab) C99HM3N7Na058 [M + H + ] theoretical 2380.8, found 2380.5. -95- (90) 1324607 MMm: Example 4 5 Synthesis of Compound 7 8 At 4. (: A cold aqueous solution of Compound 1 (20 mg) was added to the cooling

In a Dowex-5 0Wx8 (H') column (φ ο" cm x 5 cm), the dissolved aqueous solution was freeze-dried. The obtained lyophilizate was dissolved in 4 liters of cold water, and an aqueous ChCOs solution (2.5 mg n ml) was added thereto, and the adjusted value was 5 to 6 Å, and then the 6 aqueous solution was dried in a cold bed. The cold-dried Fmoc-disialyl sugar chain reagent was dissolved in Dry DMF (1.3 ml), added with decyl bromide (5.1 μl), and allowed to stand at room temperature under argon atmosphere. Mix * $ small • hour. After confirming the completion of the reaction by TLC, the reaction solution was cooled to 〇 〇c, and 10 ml of diethyl hydrazine was added to precipitate a substance of interest. Filter it with filter paper. Distilled water was added to the residual object to dissolve it in the liquid, and then the concentration was continued under reduced pressure. The obtained residue was poured into a 〇DS column (φ 1.6 cm X 14 cm, an eluent: h2O-40o/〇 MeOH aqueous solution) to obtain a purified compound 7 8 (1 8 · 2 mg, yield 8 5 %). The physical properties of the obtained compound 7.8 are as follows. ^-NMR (30 ° C) ® 7.90 (d, 2H, Fmoc), 7.70 (d, 2H, Fmoc), 7.53 -7.40 (m, 9H,

Bn, Fmoc), 5.36 (d, 2H, J=11.6 Hz, CH2), 5.30 (d, 2H, J=11.6 Hz, CH2), 5.12 (s, 1H, Man4-H,), 4.99 (d, 1H , J = 9.7 Hz, GlcNAcl-HO, 4.93 (s, 1H, Man4'-H,), 4.75 (s, 1H, Man3-H,), 4,57 (m, 3H, GlcNAc2-Hi, GlcNAc5, 4.32 (d, 2H, Gal6,6'-Hj), 4.24 (d, 1 Η,M an 3 - H 2 ),4.1 8 ( d, 1H, Man4'-H2), 4.10 (1H, d, Man4-H2 ), 2.72 (bd, 1H, Asn-β CH), 2.67 (dd, 2H, NeuAc7, 7'-H3eq)5 2.51 (bdd, 1H, •96· 1324607 (91)

Asn-β C Η), 2.06 (s, 3 Η, Ac), 2.03, 2.01 (each s, each 6H, Ac χ2), 1.89 (s, 3H, Ac), 1.83 (2H, dd, J= 12.2, 12.2 Hz, NeuAc7, 7.-H3ax); HRMS C117H165N8Na2066 [M + Na + ] Theoretical value 2783.9597, measured value 2783.9501 Industrial Applicability According to the present invention, various kinds of desired sugar chain structures can be produced very easily and in large quantities. Sugar chain. Therefore, it can be expected to be utilized for therapeutic drugs such as cancer, inflammatory diseases, and influenza. Among them, the sugar chain aspartame derivative and the sugar chain aspartame produced according to the present invention have no risk of mixing toxic components in the step, and have excellent safety -97-

Claims (1)

1324607 Announcement 丨 ' 091121856 Patent Application Chinese Patent Application Scope Replacement (January 99) Pickup, Patent Scope 1. A method for producing a sugar chain aspartate derivative derived from a sugar chain aspartame And comprising: (a) a step of obtaining a mixture of sugar chain aspartame derivatives, which is attached to the sugar chain aspartate contained in a mixture of one or more sugar chain aspartame, Introducing a mercaptomethoxycarbonyl (Fmoc) group and a third butyloxycarbonyl (Boc) group; and (b) a step of isolating each of the sugar chain aspartate derivatives, which is derived from the sugar chain aspartate The sugar chain aspartate derivative contained in the mixture of the mixture or the sugar chain aspartate derivative is hydrolyzed to obtain a mixture, which is then subjected to chromatography analysis. 2. The method for producing a sugar chain aspartame derivative according to the first aspect of the invention, further comprising: (b') using a sugar hydrolysate hydrolysis step (b) to separate the sugar chain aspartate The step of the derivative. 3. The method for producing an indole chain aspartame derivative according to the first or second aspect of the invention, wherein the mixture comprising one or more sugar chain aspartames comprises the following Compounds and/or compounds having more than one sugar residue deleted on the following compounds: 80874-990129.DOC I3246Q7 _ ' IWSiS Van SSk X The method for producing a sugar chain aspartame derivative according to any one of claims 1 to 3, wherein the step (a) is carried out on the non-reducing end One or two kinds of sialic acid residues are introduced into the Fmoc group on the sugar chain aspartate contained in a mixture of sugar chain aspartame in 80874-990129.DOC 1324607 SiliSaiM, and at the same time, in the sialic acid residue A benzyl group is introduced into the group to prepare a mixture of sugar chain aspartame derivatives. A method for producing a sugar chain aspartame, which comprises: (a) a step of obtaining a mixture of sugar chain aspartame derivatives, which comprises one or more sugar chain aspartame a step of introducing the thiol oxycarbonyl (Fmoc) group and the third butyl oxycarbonyl (Boc) group to the sugar chain aspartate contained in the mixture; (b) the step of isolating each sugar chain aspartate derivative Is a mixture of the sugar chain aspartate derivative mixture or the sugar chain aspartate derivative contained in the sugar chain aspartate derivative mixture, to obtain a mixture, and then to be used for coloring a layer analysis; and (c) a step of obtaining a sugar chain aspartate which removes the protecting group of the sugar chain aspartate derivative isolated in the step (b). 6. The method for producing a sugar chain aspartame described in claim 5, further comprising: (b1) using a sugar chain aspartate derivative isolated by the hydrolysis hydrolyzing enzyme hydrolysis step (b) And/or (c1) the step of hydrolyzing the sugar chain asparagine obtained in the step (c) using a glycolytic enzyme. 7. The method for producing a sugar chain aspartame according to the fifth or sixth aspect of the invention, wherein the mixture comprising one or more sugar chain aspartames comprises the following compounds and / or compounds with more than one sugar residue on the following compounds: 80874-990129.DOC 1324607 A 'well please specialize in killing ϊ S The method for producing a sugar chain aspartame described in any one of the items 5 to 7 wherein the step (a) includes saliva on the non-reducing end. One or more sugars of acid residues 80874-990129.DOC 1324607 The sugar chain aspartate contained in the mixture of the patent application range of asparagine is introduced into the Fmoc group while being in saliva. A mixture of the sugar chain aspartate derivative is prepared by introducing an acid residue onto the base. A method for producing a sugar chain, comprising: (a) a step of obtaining a mixture of sugar chain aspartame derivatives, which is contained in a mixture comprising one or more sugar chain aspartame a sugar chain aspartate, a mercapto oxime oxycarbonyl (Fmoc) group and a third butyl oxycarbonyl (Bo c) group; (b) a step of isolating each sugar chain aspartate derivative, which is The sugar chain aspartate derivative mixture or the sugar chain aspartate derivative contained in the sugar chain aspartate derivative mixture is hydrolyzed to obtain a mixture, which is then subjected to chromatograph analysis; c) a step of obtaining a sugar chain aspartate, which is a step of removing the protecting group of the sugar chain aspartate derivative isolated in the step (b); and (d) a step of obtaining a sugar chain, which is a removal step (c) The aspartate residue of the obtained sugar chain aspartate. 10. The method for producing a sugar chain according to claim 9, further comprising: (b) a step of hydrolyzing the sugar chain aspartate derivative separated by the step (b); and / or (c') a step of hydrolyzing the sugar chain asparagine obtained by the step (c) using a glycolytic enzyme; and/or (d') using the sugar hydrolyzing enzyme to hydrolyze the sugar chain obtained in the step (d) . 11. The method of manufacturing a sugar chain according to claim 9 or claim 10, wherein the mixture comprising one or more sugar chain aspartame contains the following Compounds and/or compounds having one or more sugar residues deleted on the following compounds: 80874-990129.DOC 1324607 Scope of application for patent continuation of the end of the chain 醯 醯 醯 冬 冬 冬 冬 载 载 载 载 载 载 非 非 非 非 非 非 非 非 非 非 非 非 非 非 非 非 非 第 第 第 第 第 第 第 第 第 第 第 第 第In this case, the base of the syllabary is surrounded by its acidity, and the liquefaction method has a prescription for the application of the product or the E. The substance is mixed with acid and has a liquid mixed amine containing salivation. In the middle of winter, the derivative is the same as the amine, and the chain 1 is mixed with Cλ winter sugar R \ο^. The sugar-based indole is introduced into the chain-conducting sugar, and the amine is obtained, and the formula is as follows. (wherein, Asn is aspartame, and R1 and R2 may be the same or different and 80874-990129.DOC 132460.7 ϋ ϋ Patent Range Continued Page 1 Except in the case of). 14. A sugar chain aspartame derivative having the general formula: Rx RY (where Rx and RY are one of 80874-990129.DOC 1324607 OH Or ). 15. A sugar chain aspartame, which has the general formula: 80874-990129.DOC 9· 1324607 (wherein, Asn is aspartame, R3 and R4 may be the same or different, but 80874-990129.DOC -10- 1324607 for Η or ί the case of applying for a patent coverage; and (3) R3 is Η, and R4 is 16. except for the case of a sugar chain). General (where R5 and R6 may be the same or different, but Η, 80874-990129.DOC -11 - 1324607 , but (1) R5 and R6 are the same; (2) R5 and R6, one of which is NHAc 80874-990l29.DOC -12-