JP5680576B2 - Method for producing 11-saccharide sialyl-oligosaccharide asparagine - Google Patents

Description

  The present invention relates to a method for producing an 11-saccharide sialyl oligosaccharide asparagine. The present invention also relates to a method for producing an 11-saccharide sialyl oligosaccharide asparagine into which a fat-soluble protecting group is introduced.

In recent years, sugar chains have attracted attention as biomolecules in addition to nucleic acids and proteins.
Membrane proteins and sugar chains existing outside the cell are considered to have functions related to recognition and interaction between cells. And it is thought that the change in recognition and interaction between cells is a cause of causing cancer, chronic diseases, infectious diseases, and aging.

Many sugar chains are present in vivo as glycoproteins and glycolipids, and are thought to bear physiological functions through recognition and interaction between cells.
In vivo, sugar chains not only have a variety of structures, but also exist in minute amounts and often exist in association with proteins, etc., so purification generally requires a number of steps. Many sugar chains are known to be difficult to extract and purify.

As research in the field of glycochemistry has progressed in recent years, research on enzymes that replace sugar chains of glycoproteins with yeast-type sugar chains with human-type sugar chains is accelerating. It is now possible to substitute sugar chains. According to this method, it is possible to supply a large amount of a glycoprotein pharmaceutical having a human-type sugar chain at a low cost compared to producing a glycoprotein having a human-type sugar chain using animal cells.
For example, erythropoietin having a uniform human sugar chain can be produced by replacing the yeast sugar chain of erythropoietin expressed in yeast with a human sugar chain.
The glycoprotein pharmaceutical obtained by such a method retains its homogeneity as a pharmaceutical because its sugar chain structure is human and identical. As described above, in the production of antibody drugs and physiologically active proteins, a method for supplying human-type sugar chains and replacing yeast-type sugar chains of glycoproteins expressed in yeast with human-type sugar chains will be very important in the future. It is thought that it can be an effective method.

However, since a method for supplying a large amount of human-type sugar chain has not yet been established, establishment of this method is desired.
In recent years, egg yolk has attracted attention as a source of human sugar chains. Since egg yolk mainly contains the same double-chain N-linked complex sugar chains as human sugar chains, if the sugar chains can be efficiently extracted in large quantities, egg yolk is used as a human source. Type sugar chains can be supplied.

For example, Patent Document 1 discloses preparing an 11-saccharide sialyl oligosaccharide peptide from hen egg-derived defatted egg yolk (powder). Patent Document 1 discloses that sialic acid derivatives to which amino acids and peptides are not bound can be obtained by adding almonds or apricot seeds to hen egg-derived defatted egg yolk.
Non-Patent Document 1 discloses that a sugar chain peptide (SGP: sialyl glycopeptide) extracted from a soluble fraction of chicken eggs is obtained. This SGP is a compound in which a peptide residue of a peptide chain consisting of 6 amino acid residues is bonded to the reducing end of a complex type sugar chain consisting of 11 sugar residues consisting of a human type sugar chain.
Patent Document 2 discloses a method for producing a sialic acid-containing oligosaccharide by extracting it with water or a salt solution from defatted egg yolk.

  Patent Document 3 discloses a method for producing a sugar chain asparagine derivative by introducing a fat-soluble protecting group (Fmoc group or Boc group) into an amino group.

International Publication No. 96/02255 Pamphlet Japanese Patent Application Laid-Open No. 8-99988 International Publication No. 2004/070046 Pamphlet

Akira Seko, Mamoru Koketsu, Masakazu Nishizono, Yuko Enoki, Hisham R. Ibrahim, Lekh Raj Juneja, Mujo Kim, Takehiko Yamamoto, Biochimica et Biophysica Acta, 1997, Vol.1335, p.23-32

  The sugar chain asparagine derivative prepared by the method disclosed in Patent Document 3 includes 1) a step of producing a glycopeptide mixture from defatted egg yolk using a proteolytic enzyme, and 2) a sugar chain asparagine using a peptide digestion enzyme. A step of producing a mixture, 3) a step of producing a sugar chain asparagine derivative mixture by introducing a fat-soluble protective group into the sugar chain asparagine in the sugar chain asparagine mixture, and 4) each of the sugar chain asparagine derivative mixtures subjected to chromatography. It has been obtained through extremely complicated steps including a step of separating a sugar chain asparagine derivative.

  The problem to be solved by the present invention is to provide a method for producing an 11-saccharide sialyl-oligosaccharide asparagine with higher purity and yield in a simpler manner than bird egg defatted egg yolk.

  As a result of intensive studies to solve the above problems, the inventors of the present invention, in a method for producing 11 sugar sialyl oligosaccharide asparagine using bird egg defatted egg yolk as a raw material, first, 11 egg sialyl oligosaccharide from bird egg defatted egg yolk. The present invention was completed by finding that the above-mentioned problems can be solved by a production method for obtaining an asparagine mixture and then desalting a precipitate obtained by alcohol precipitation of an 11-saccharide sialyl oligosaccharide asparagine mixture.

That is, the present invention is as follows.
[1]
A method for producing an 11-saccharide sialyl-oligosaccharide asparagine represented by the following formula 1 from a defatted egg yolk:
Obtaining an 11-saccharide sialyl-oligosaccharide asparagine mixture from a defatted egg yolk, then subjecting the mixture to alcohol precipitation to obtain a sugar chain asparagine precipitate, and a desalting step of desalting the sugar chain asparagine precipitate. A method for producing an 11-saccharide sialyl-oligosaccharide asparagine.
Formula 1:
[2]
A method for producing an 11-saccharide sialyl-oligosaccharide asparagine represented by the following formula 1 from a defatted egg yolk:
An extraction process for extracting a glycopeptide extract by extracting avian egg defatted egg yolk with water or a salt solution;
A precipitation step of precipitating alcohol from the extract to obtain a glycopeptide precipitate;
Desalting the precipitate to obtain an 11-saccharide sialyl oligosaccharide peptide;
Degrading the glycopeptide with a peptide degrading enzyme to obtain an 11-saccharide sialyl-oligosaccharide asparagine mixture;
A method for producing an 11-saccharide sialyl-oligosaccharide asparagine comprising a precipitation step of precipitating alcohol from the mixture to obtain a sugar chain asparagine precipitate, and a desalting step of desalting the sugar chain asparagine precipitate.
Formula 1:
[3]
The production according to [1] or [2], wherein the desalting step of desalting the glycopeptide precipitate or the sugar chain asparagine precipitate is a step of holding the precipitate on a resin and then washing with water. Method.
[4]
The production method according to [3], wherein the resin is a resin for packing in reverse phase partition chromatography.
[5]
The production method according to [4], wherein the resin for packing in reverse phase partition chromatography is a chemically bonded silica gel resin.
[6]
The production method according to any one of [1] to [5], wherein the desalting includes a step of eluting with an organic solvent aqueous solution containing at least one selected from the group consisting of acetonitrile, methanol, and ethanol.
[7]
The production method according to [6], wherein the concentration of the organic solvent aqueous solution is 0.1 to 10% (v / v).
[8]
A method for producing an 11-saccharide sialyl-oligosaccharide asparagine represented by the following formula 1 from a defatted egg yolk:
Extraction process for obtaining glycopeptide extract by extracting avian egg defatted egg yolk with water,
Precipitation step of obtaining a glycopeptide precipitate by ethanol precipitation from the extract,
Desalting the precipitate with ODS resin to obtain an 11-saccharide sialyl oligosaccharide peptide;
Degrading the glycopeptide with a peptide degrading enzyme to obtain an 11-saccharide sialyl-oligosaccharide asparagine mixture;
A method for producing an 11-saccharide sialyl oligosaccharide asparagine, comprising a precipitation step of precipitating ethanol from the mixture to obtain a sugar chain asparagine precipitate, and a desalting step of desalting the sugar chain asparagine precipitate.
Formula 1:
[9]
The production method according to any one of [1] to [8], wherein the 11-saccharide sialyl oligosaccharide asparagine has a structure represented by the following formula 2.
Formula 2:
[10]
A step of introducing a fat-soluble protecting group into an amino group derived from asparagine of the 11-saccharide sialyl-oligosaccharide asparagine obtained by the production method according to any one of [1] to [9], and an alcohol precipitation step A method for producing an 11-saccharide sialyl oligosaccharide asparagine into which a fat-soluble protecting group is introduced.
[11]
The lipophilic protecting group is a 9-fluorenylmethyloxycarbonyl group, benzyloxycarbonyl group, tert-butoxycarbonyl group, trityl group, 2,2,2-trichloroethoxycarbonyl group, allyloxycarbonyl group, acetyl group, The production method according to [10], which is selected from the group consisting of a phthaloyl group, a p-toluenesulfonyl group, and a 2-nitrobenzenesulfonyl group.

  According to the present invention, an 11-saccharide sialyl oligosaccharide asparagine can be easily produced with high purity and high yield.

The HPLC chart of the 11 sugar sialyl oligosaccharide peptide manufactured in Example 1 is shown. 1 shows a ¹ H-NMR chart of an 11-saccharide sialyl oligosaccharide peptide produced in Example 1. The HPLC chart of the 11 sugar sialyl oligosaccharide peptide manufactured by re-elution using ODS resin in Example 1 is shown. The HPLC chart of the 11 sugar sialyl oligosaccharide asparagine manufactured in Example 1 is shown. The HPLC chart of the 11 sugar sialyl oligosaccharide asparagine manufactured in Example 2 is shown. The HPLC chart of Fmoc11 sugar sialyl oligosaccharide asparagine manufactured in Example 3 is shown.

  Hereinafter, a mode for carrying out the present invention (hereinafter referred to as “the present embodiment”) will be described in detail. In addition, this invention is not limited to the following embodiment, It can implement by changing variously within the range of the summary.

  The method for producing the 11-saccharide sialyl-oligosaccharide asparagine (hereinafter sometimes simply referred to as “sugar chain asparagine”) of the present embodiment is obtained by obtaining a mixture of 11-saccharide sialyl-oligosaccharide asparagine from avian egg defatted egg yolk. And a precipitation step of precipitating alcohol from the mixture to obtain a sugar chain asparagine precipitate, and a desalting step of desalting the sugar chain asparagine precipitate.

In the present embodiment, the 11-saccharide sialyl oligosaccharide asparagine means a compound represented by the following formula 1 in which asparagine is bonded to the reducing end of a complex type sugar chain composed of 11 sugar residues.
Formula 1:

According to the production method of the present embodiment, the 11-saccharide sialyl oligosaccharide asparagine can be produced more easily and preferably at a lower cost than avian egg defatted egg yolk.
In addition, in a preferable aspect of the present embodiment, the 11-saccharide sialyl oligosaccharide asparagine is obtained with a high purity of 90% or more, preferably 93%, more preferably 95% or more, and yield by a simple process on an industrial scale. Can be manufactured well.

In the present embodiment, an 11-saccharide sialyl-oligosaccharide peptide mixture is obtained from bird egg defatted egg yolk, and an 11-saccharide sialyl-oligosaccharide asparagine mixture can be obtained by performing the following steps (hereinafter, simply “ The “11 saccharide sialyl oligosaccharide peptide” may be referred to as “glycopeptide”.
As such a process, an extraction step of extracting defatted avian egg yolk with water or a salt solution to obtain a glycopeptide extract, a precipitation step of precipitating alcohol from the extract to obtain a glycopeptide precipitate, and removing the precipitate Salting to obtain an 11-saccharide sialyl-oligosaccharide peptide and degrading the glycopeptide with a peptide-degrading enzyme to obtain an 11-saccharide sialyl-oligosaccharide asparagine mixture.

Extraction of avian egg defatted egg yolk with water or a salt solution to obtain a glycopeptide extract is a suspension of a bird egg defatted egg yolk in water or a salt solution to extract a mixture of glycopeptides, etc. This is a step of obtaining a glycopeptide extract which is a crudely purified product.
Examples of glycopeptides contained in the glycopeptide extract include compounds represented by the following formula 3.
Formula 3:

  A peptide chain consisting of 6 amino acid residues (Lys-Val-Ala-Asn-Lys-Thr) is bound to the reducing end of the sugar chain of the glycopeptide represented by Formula 3 (SEQ ID NO: 1).

The bird egg defatted egg yolk is not particularly limited, and examples thereof include commercially available defatted egg yolk and defatted egg yolk prepared from a bird egg.
Bird eggs are not particularly limited, but examples include eggs such as chickens, quail, ducks, ducks, ostriches, and pigeons, and the amount of glycopeptide contained in egg yolk is high. Eggs that are eggs are preferred.
Avian egg defatted egg yolk can be obtained by degreasing the whole egg or egg yolk of an avian egg with an organic solvent.
The bird egg may be a raw egg, a dried egg powder obtained by drying, and preferably used are egg yolk and egg yolk powder.

The method of degreasing is not particularly limited, and examples thereof include a method of adding an organic solvent to a bird egg to separate a precipitate from an organic solvent layer.
Although it does not specifically limit as an organic solvent used for a degreasing process, For example, acetone, methanol, ethanol, 2-propanol etc. are mentioned, One type of solvent may be used as an organic solvent, A mixed solvent of two kinds of solvents may be used.
In the degreasing treatment, the amount of the organic solvent to be added to the bird egg is not particularly limited, but the degreasing treatment can be performed by using 1 to 5 times as much organic solvent by weight as the bird egg. it can.
The degreasing treatment is not particularly limited, but can be performed at a temperature of 0 to 25 ° C.

After adding an organic solvent to an avian egg, the fat contained in the avian egg can be removed by the organic solvent by thoroughly stirring the organic solvent and the avian egg.
Although it does not specifically limit as a method to isolate | separate an organic solvent and a deposit, You may centrifuge at 2,000-10,000 rpm for 5 to 30 minutes.
The degreasing treatment using an organic solvent is not particularly limited, but is preferably performed 2 to 6 times.

There is no particular limitation on the method of extracting glycopeptide from avian egg defatted egg yolk by adding water or a salt solution to the defatted egg yolk. Although it does not specifically limit as a salt solution used in an extraction process, For example, sodium chloride aqueous solution, a phosphate buffer, etc. are mentioned, As a salt solution, 1 type of salt solutions may be used. You may use the mixed salt solution of the salt solution of a seed | species or more.
The concentration of the salt solution is not particularly limited, but is 0.0001 to 2.0% (w / v). Although it does not specifically limit as quantity of the water or salt solution added to a bird egg defatted egg yolk, By using water or a salt solution 0.1-50 times by mass with respect to a bird egg defatted egg yolk Extraction of glycopeptides can be performed.
Extraction of the glycopeptide is not particularly limited, but can be performed at a temperature of 4 to 25 ° C.

After adding water or salt solution to bird egg defatted egg yolk, the avian egg defatted egg yolk and water or salt solution are well stirred to extract the glycopeptide contained in the bird egg defatted egg yolk with water or salt solution it can.
A method for separating an extract containing a glycopeptide extracted with water or a salt solution (glycopeptide extract) and avian egg defatted egg yolk is not particularly limited, but is 2,000 to 10,000 rpm. May be centrifuged for 5 to 30 minutes.
The extraction treatment using water or a salt solution is not particularly limited, but is preferably performed 2 to 6 times, and more preferably 2 to 4 times.
The extraction step is preferably performed using water.

The precipitation step of precipitating alcohol from a glycopeptide extract to obtain a glycopeptide precipitate is to concentrate the extract of glycopeptide by adding the extract containing the glycopeptide obtained in the extraction step to alcohol. In addition to this, it is a step of obtaining a glycopeptide having an improved degree of purification as a precipitate (glycopeptide precipitate). In the precipitation step, a glycopeptide precipitate may be precipitated as a crude product by adding alcohol to the extract.
In the present embodiment, the alcohol is not particularly limited, and examples thereof include a solvent selected from the group consisting of alcohols such as methanol, ethanol, propanol, butanol, pentanol, ethylene glycol, and glycerin. . One solvent may be used, or a mixed solvent of two or more solvents may be used. In particular, it is preferably an alcohol having 1 to 5 carbon atoms. In the precipitation step, when the water-soluble organic solvent is an alcohol, the precipitation step adds the glycopeptide extract to the alcohol to precipitate the glycopeptide. Thus, a glycopeptide precipitate is obtained (hereinafter sometimes referred to as “alcohol precipitation step”), and the glycopeptide extract obtained in the extraction step is added to alcohol to obtain a glycopeptide precipitate. This is an alcohol precipitation step that not only concentrates the extract but also obtains a glycopeptide with improved purity as a precipitate.

The amount of alcohol used in the alcohol precipitation step is not particularly limited, but the glycopeptide can be extracted by using 2 to 20 times as much alcohol as the mass of the extract. Moreover, as alcohol used for an alcohol precipitation process, C1-C5 alcohol is preferable and C1-C3 alcohol is more preferable. Specific examples of the alcohol having 1 to 3 carbon atoms include methanol, ethanol, and 2-propanol (isopropanol). Methanol and ethanol are preferable, and ethanol is particularly preferable. When a glycopeptide is used for medical use or pharmaceutical use, it is preferable to use ethanol as the alcohol used in the alcohol precipitation step.
In the present embodiment, as the alcohol used in the alcohol precipitation step, one kind of alcohol may be used, a mixed solvent of two or more kinds of alcohols may be used, or a mixed solvent of alcohol and another solvent is used. May be.
When the alcohol solvent is a mixture, for example, a mixed solvent obtained by adding 0.01 to 50% of methanol or 2-propanol to ethanol may be used. Moreover, you may use the mixed solvent which added acetone to 50% of acetone, acetonitrile, or diethyl ether with respect to ethanol.

  Extraction of the glycopeptide is not particularly limited, but can be performed at a temperature of 4 to 25 ° C.

  The glycopeptide extract obtained in the previous extraction step used in the alcohol precipitation step can be made into a clear extract by filtering the extract before the precipitation step. You may use the concentrate of the extract obtained by concentrating.

In the alcohol precipitation step, the method for separating the glycopeptide is not particularly limited, but may be centrifuged at 2,000 to 10,000 rpm for 5 to 30 minutes and left at 4 to 25 ° C. May be separated.
A more purified glycopeptide can be obtained by dissolving the obtained glycopeptide in water or a salt solution and performing an alcohol precipitation step again. Since the glycopeptide does not dissolve in alcohol, the alcohol precipitation step can be repeated as necessary to remove impurities. By repeating the alcohol precipitation step, a more purified glycopeptide can be obtained, and precipitation of the glycopeptide is not particularly limited, but is preferably performed 2 to 6 times, and 2 to 4 times. It is more preferable.

The desalting step of desalting the glycopeptide precipitate is a step of removing a salt from a precipitate (glycopeptide precipitate) as a crude product containing the glycopeptide obtained in the precipitation step.
The desalting step can be performed by various known methods known as desalting methods and is not particularly limited. The desalting step is not particularly limited, and desalting can be performed using an ion exchange resin, an ion exchange membrane, gel filtration, a dialysis membrane, an ultrafiltration membrane, a reverse osmosis membrane, or the like. As the desalting step, for example, the precipitate obtained in the precipitation step can be retained on the resin, and then desalted by washing with water.
In the present embodiment, the 11-saccharide sialyl oligosaccharide peptide can be easily produced on an industrial scale by desalting the precipitate obtained by performing the extraction step and the precipitation step described above. .
In the conventional method as described in Patent Document 1, extraction from chicken egg-derived defatted egg yolk (powder) with water, concentration with a reverse osmosis membrane, adsorption onto an anion exchange resin, desalting, and using an aqueous NaCl solution It is necessary to perform elution, concentration, and a desalting step again, and this cannot be said to be a simple method for carrying out on an industrial scale.

The precipitate can be held on the resin by a holding method using a known binding mode such as adsorption or loading. Moreover, the precipitate should just be hold | maintained to such an extent that a precipitate does not flow out with a washing | cleaning liquid, when wash | cleaning with water.
The resin is not particularly limited, and examples thereof include a resin for packing in reverse phase partition chromatography. The resin for packing in reverse phase partition chromatography is a resin represented by silica gel or polymer. Meaning, not particularly limited, for example, poly (styrene / divinylbenzene) polymer gel resin, polystyrene-divinylbenzene resin, polyhydroxymethacrylate resin, styrene vinylbenzene copolymer resin, polyvinyl alcohol resin, polystyrene resin , Polymethacrylate resin, chemically bonded silica gel resin and the like.
The chemically bonded silica gel resin is not particularly limited, and examples thereof include a resin produced by reacting a porous silica gel with a silane coupling agent such as dimethyloctadecylchlorosilane. By using different silylating agents in the above method, dimethyloctadecyl, octadecyl (C18), trimethyloctadecyl, dimethyloctyl, octyl (C8), butyl, ethyl, methyl, phenyl, cyanopropyl, aminopropyl groups are selected. And a resin obtained by chemically bonding a group to be bonded. Alternatively, a resin obtained by bonding a docosyl (C20) group having 22 carbon atoms and a triacontyl (C30) group having 30 carbon atoms may be used.
In the present embodiment, preferred chemical bond type silica gel resin includes octadecyl group-bonded silica gel resin (ODS resin) in which octadecyl group is supported using silica gel as a base material.

The desalting step preferably includes a step of eluting the glycopeptide (11-saccharide sialyl oligosaccharide peptide) retained in the resin after desalting with an organic solvent aqueous solution, and after desalting in the desalting step, By performing an elution step of eluting the glycopeptide with an aqueous solvent solution, the purity of the 11-saccharide sialyl oligosaccharide peptide can be improved. In a more preferred embodiment, the purity of the 11-saccharide sialyl oligosaccharide peptide can be 90% or higher, preferably 93%, more preferably 95% or higher. For example, the purity of the glycopeptide can be further improved by repeating the desalting step (elution step) again. Improving the purity of a glycopeptide is, for example, using a glycosyltransferase such as Endo-M to transfer a sugar chain part of the glycopeptide obtained in this embodiment to another sugar chain or a sugar chain derivative. (Including the case of transferring to a sugar chain in a peptide chain) is more preferable because interference reaction due to impurities is reduced and the reaction efficiency of the target transfer reaction is improved.
The organic solvent used in the elution step contains, for example, at least one selected from the group consisting of acetonitrile, methanol, and ethanol, and the concentration of the organic solvent aqueous solution is 0.1 as the concentration of the organic solvent in the aqueous solution. It is preferably ˜20% (v / v), more preferably 0.1 to 10% (v / v).

  Although a desalting process or an elution process is not specifically limited, it can be performed at the temperature of 4-25 degreeC.

As the desalting step, it is possible to desalinate from the precipitate obtained in the precipitation step by using a separation membrane in addition to the desalting step using a resin.
As such a desalting step, for example, the desalting of the precipitate can be performed by using an ultrafiltration membrane or a reverse osmosis membrane.
The membrane used in the desalting step is not particularly limited, and examples thereof include polyacrylonitrile, polysulfone, polyether sulfone, polyvinylidene fluoride, polytetrafluoroethylene, aromatic polyamide, cellulose acetate, and polyvinyl alcohol. Examples thereof include a flat membrane or a hollow fiber membrane, a spiral membrane or a tubular membrane as a constituent substrate. The membrane can be further desalted with an ion exchange resin, ion exchange membrane, gel filtration, dialysis membrane, ultrafiltration membrane or reverse osmosis membrane.

  In the present embodiment, as a method for purifying a glycopeptide, a known purification method such as a peptide, a carbohydrate, or a glycopeptide can be used, but it is not particularly limited. Examples thereof include normal phase chromatography, reverse phase chromatography, ion exchange chromatography, affinity chromatography, and size exclusion chromatography. The carrier used for the reverse phase chromatography is not particularly limited, and examples thereof include those obtained by immobilizing octadecyl, octyl, phenyl, cyanopropyl, methyl, etc. on the filler surface using silica as a base material. Among them, ODS resin and the like can be mentioned.

The purification method by column chromatography using a silica gel resin filler such as ODS resin may include a step of desalting a salt contained in the glycopeptide obtained by precipitation, and the glycopeptide is dissolved in an organic solvent aqueous solution. May be included.
The glycopeptide can be desalted by washing the resin with 1 to 50 times as much water by mass relative to the silica gel resin to which the glycopeptide has been added and retained. The desalting step may include a step of holding the glycopeptide precipitate in a resin, preferably an ODS resin, washing with water, and then eluting the 11-saccharide sialyl oligosaccharide peptide with an organic solvent aqueous solution.

After desalting, the glycopeptide can be obtained by elution with an organic solvent aqueous solution. In a more preferred embodiment, the purity of the 11-saccharide sialyl oligosaccharide peptide can be 90% or higher, preferably 93%, more preferably 95% or higher, and still more preferably 99% or higher.
A glycopeptide can be obtained by elution from a resin using an organic solvent aqueous solution that is 1 to 50 times the resin in mass with respect to a silica gel resin such as an ODS resin that retains the glycopeptide by adding the glycopeptide. it can.

The organic solvent aqueous solution is not particularly limited, and examples thereof include an aqueous solution of at least one organic solvent selected from the group consisting of acetonitrile, methanol, and ethanol.
Acetonitrile is preferable as the organic solvent aqueous solution. In particular, by performing the elution step with an acetonitrile aqueous solution, in the elution step of the glycopeptide in which the terminal sialic acid, which is one of the impurities, is partially cleaved, and the desired glycopeptide. The separation purity is remarkably improved, and a glycopeptide can be obtained with a purity of 95% or more.
The concentration of the organic solvent aqueous solution is preferably 0.1 to 20% (v / v), more preferably 0.5 to 20% (v / v), still more preferably 10% (v / v). Hereinafter, it is more preferably 5% (v / v) or less. The concentration of the organic solvent aqueous solution is preferably 0.5 to 3% (v / v).

In the conventional method as described in Patent Document 1, in the elution operation from a resin, for example, when an anion exchange resin or the like is used, the elution operation using a salt solution or an acidic solution is performed after adsorbing to the resin. Is required. However, elution with an acidic solution is not preferable because terminal sialic acid may be detached from the glycopeptide. In addition, in the case of elution with a salt solution, it is necessary to perform a desalting treatment from the eluate again, which increases the number of steps in mass production. In the present embodiment, the elution step is preferably performed with an organic solvent aqueous solution, so that the desalting operation of the eluate is not required and the number of steps can be reduced, which is suitable for mass production. In addition, the method according to the present embodiment is advantageous in that the pH does not change when the eluate is concentrated and the decomposition of the sugar chain portion of the glycopeptide is less than the methods disclosed so far.
In the step of eluting the glycopeptide with the organic solvent aqueous solution, the elution may be performed by gradually grading the organic solvent aqueous solution as the eluent from water.

The eluted glycopeptide can be obtained as a powdered glycopeptide by concentration under reduced pressure of an organic solvent aqueous solution and drying.
In the present embodiment, the obtained glycopeptide may be further purified by performing an alcohol precipitation step again. In addition, by adsorbing the glycopeptide again to silica gel resin such as ODS resin, and elution from the resin again using an organic solvent aqueous solution 1 to 50 times the mass of the resin, higher purity (99% or more) Can be obtained.

The step of degrading an 11-saccharide sialyl oligosaccharide peptide with a peptide-degrading enzyme to obtain an 11-saccharide sialyl-oligosaccharide asparagine mixture is an enzyme containing an 11-saccharide sialyl oligosaccharide asparagine by selectively hydrolyzing the peptide portion of the glycopeptide. This is a step of obtaining a mixture of degradation products (11 sugar sialyl oligosaccharide asparagine mixture). Usually, an asparagine mixture can be obtained as an enzyme reaction solution containing a sugar chain asparagine.
In this step, the 11-saccharide sialyl oligosaccharide peptide represented by the above formula 4 is enzymatically decomposed by a peptide degrading enzyme and converted to the 11-saccharide sialyl oligosaccharide asparagine represented by the above formula 1. In general, the stereochemistry of the sugar chain asparagine is enzymatically degraded in such a way that the stereochemistry of the glycopeptide is retained.

As the peptide degrading enzyme, for example, protease such as general carboxypeptidase and aminopeptidase such as pronase (manufactured by Wako Pure Chemical Industries), actinase-E (manufactured by Kaken Pharmaceutical Co., Ltd.), orientase and the like can be used.
Examples of the protease include endo-type protease having an optimum pH of 7 to 8, for example, proteases produced by Aspergillus genus, Bacillus genus and the like.
Specific examples include proteases for food additives such as orientase ONS, 90N (manufactured by Hankyu Bio Inc.), protease N (manufactured by Amano Pharmaceutical Co., Ltd.), AO protease (manufactured by Shengshin Pharmaceutical Co., Ltd.), and other sources. And enzymes such as actinase E, chymotrypsin, and trypsin.
The enzymatic degradation reaction can proceed to the 11-saccharide sialyl-oligosaccharide asparagine by carrying out at the optimum pH, temperature and ionic strength of each protease.
As the decomposition reaction conditions, it is preferable to perform the enzyme decomposition reaction at pH 7 to 8, temperature 30 to 50 ° C., and ionic strength 0.1 to 1.0.
In addition, the amount of the peptide degrading enzyme in the enzyme decomposing reaction can be efficiently degraded to 11 sugar sialyl oligosaccharide asparagine by adding 0.03 to 20% (w / w) to defatted egg yolk. .

Next, the sugar chain asparagine mixture is separated in the precipitation process of sugar chain asparagine using alcohol.
The step of precipitating alcohol from the 11-saccharide sialyl-oligosaccharide asparagine mixture to obtain the sugar chain asparagine precipitate is performed by adding the mixture containing the sugar chain asparagine obtained in the enzyme reaction step (sugar chain asparagine mixture) to the alcohol. This is a step of not only concentrating a mixture of sugar chain asparagine but also obtaining sugar chain asparagine having improved purity as a precipitate (sugar chain asparagine precipitate).
In this precipitation step, sugar chain asparagine may be precipitated as a crude product by adding alcohol to the mixture. In this step, sugar chain asparagine and enzyme are mainly obtained as a precipitation product.

In the present embodiment, this precipitation step can be performed in the same manner as the method described as the precipitation step described above, wherein the glycopeptide precipitate is obtained by alcohol precipitation from the glycopeptide extract. Moreover, you may perform this precipitation process as said alcohol precipitation process.
The alcohol in the present embodiment is not particularly limited, and examples thereof include a solvent selected from the group consisting of alcohols such as methanol, ethanol, propanol, butanol, pentanol, ethylene glycol, and glycerin. . One solvent may be used, or a mixed solvent of two or more solvents may be used. In particular, it is preferably an alcohol having 1 to 5 carbon atoms. In the precipitation step, when the water-soluble organic solvent is an alcohol, the precipitation step includes adding a sugar chain asparagine extract to the alcohol to form a sugar chain asparagine. Of the sugar chain asparagine by adding the extract containing the sugar chain asparagine obtained in the extraction step to the alcohol (hereinafter, sometimes referred to as “alcohol precipitation step”). This is an alcohol precipitation step that not only concentrates the mixed solution but also obtains sugar chain asparagine with improved purity as a precipitate.

  The amount of alcohol used in the alcohol precipitation step is not particularly limited, but sugar chain asparagine can be extracted by using 2 to 20 times as much alcohol by mass as the enzyme reaction solution. . Moreover, as alcohol used for an alcohol precipitation process, C1-C5 alcohol is preferable and C1-C3 alcohol is more preferable. Specific examples of the alcohol having 1 to 3 carbon atoms include methanol, ethanol, and 2-propanol (isopropanol). Methanol and ethanol are preferable, and ethanol is particularly preferable. When sugar chain asparagine is used for medical use or pharmaceutical use, it is preferable to use ethanol as the alcohol used in the alcohol precipitation step. When the alcohol solvent is a mixture, for example, a mixed solvent obtained by adding 0.01 to 50% of methanol or 2-propanol to ethanol may be used. Moreover, you may use the mixed solvent which added acetone to 50% of acetone, acetonitrile, or diethyl ether with respect to ethanol.

  Extraction of sugar chain asparagine is not particularly limited, but can be performed at a temperature of 4 to 25 ° C.

In the alcohol precipitation step, the method for separating the sugar chain asparagine is not particularly limited, but may be centrifuged at 2,000 to 10,000 rpm for 5 to 30 minutes and left at 4 to 25 ° C. May be separated.
By dissolving the obtained sugar chain asparagine in water or a salt solution and performing an alcohol precipitation step again, a more purified sugar chain asparagine can be obtained. Since sugar chain asparagine does not dissolve in alcohol, the alcohol precipitation step can be repeated as necessary to remove impurities. By repeating the alcohol precipitation step, a more purified sugar chain asparagine can be obtained, and the precipitation of the sugar chain asparagine is not particularly limited, but it is preferably performed 2 to 6 times, and 2-4 More preferably, it is performed once.

Subsequently, the sugar chain asparagine is separated in the desalting step of the sugar chain asparagine and the mixture.
The desalting step for desalinating the sugar chain asparagine precipitate is a low-purity salt, peptide, amino acid, etc. from a precipitate (sugar chain asparagine precipitate) as a crude product containing the sugar chain asparagine obtained in the precipitation step. This is a step of removing molecular weight compounds or enzymes.
This desalting step can be performed in the same manner as the method described as the desalting step described above for desalting the glycopeptide precipitate.
The desalting step can be performed by various known methods known as desalting methods and is not particularly limited. The desalting step is not particularly limited, and desalting can be performed using an ion exchange resin, an ion exchange membrane, gel filtration, a dialysis membrane, an ultrafiltration membrane, a reverse osmosis membrane, or the like. As the desalting step, for example, the precipitate obtained in the alcohol precipitation step can be retained on the resin, and then desalted by washing with water.
Specifically, the method includes a step of desalting a salt contained in a sugar chain asparagine obtained by known chromatography, for example, gel filtration chromatography or column chromatography using a silica gel resin filler such as ODS resin. May be. In this desalting step, the sugar chain asparagine is separated.

The desalting step of the sugar chain asparagine is not particularly limited, but can be performed at a temperature of 4 to 25 ° C.
After desalting, sugar chain asparagine can be obtained by elution with an organic solvent aqueous solution. In a more preferred embodiment, the purity of the 11-saccharide sialyl oligosaccharide asparagine can be 90% or more, preferably 93%, more preferably 95% or more.

  In the present embodiment, as a method for purifying a sugar chain asparagine, a known peptide, carbohydrate, or glycopeptide purification method can be used in the same manner as the above-described method for purifying a glycopeptide. It is not particularly limited. Examples thereof include normal phase chromatography, reverse phase chromatography, ion exchange chromatography, affinity chromatography, and size exclusion chromatography. The carrier used for the reverse phase chromatography is not particularly limited, and examples thereof include those obtained by immobilizing octadecyl, octyl, phenyl, cyanopropyl, methyl, etc. on the filler surface using silica as a base material. Among them, ODS resin and the like can be mentioned.

The purification method by column chromatography using a silica gel resin filler such as ODS resin may include a step of desalting a salt contained in the sugar chain asparagine obtained by precipitation, and the sugar solution may be added by an organic solvent aqueous solution. A step of eluting chain asparagine may be included.
The sugar chain asparagine can be desalted by washing the resin with 1 to 50 times the mass of water with respect to the silica gel resin added and held with the sugar chain asparagine. The desalting step may include a step of holding the sugar chain asparagine precipitate in the resin, preferably ODS resin, washing more with water, and then eluting the 11-saccharide sialyl oligosaccharide asparagine with an organic solvent aqueous solution. .

The 11-saccharide sialyl oligosaccharide asparagine obtained by the production method in the present embodiment is an 11-saccharide sialyl oligosaccharide asparagine represented by the following formula 1.
Formula 1:
The sugar chain asparagine may have a structure represented by the following formula 2.
Formula 2:

  In the present embodiment, a fat-soluble protecting group is introduced, including a step of introducing a fat-soluble protecting group and an alcohol precipitation step, with respect to the amino group derived from asparagine of the sugar chain asparagine obtained as described above. A method for producing an 11-saccharide sialyl-oligosaccharide asparagine is also provided (hereinafter, “11-saccharide sialyl-oligosaccharide asparagine into which a fat-soluble protecting group is introduced” may be simply referred to as “sugar chain asparagine derivative”). .

  In the present embodiment, “the fat-soluble protecting group is introduced” means that the fat-soluble protecting group is bonded to the amino group derived from asparagine of the sugar chain asparagine.

  Examples of the fat-soluble protecting group include fat-soluble protecting groups selected from carbamate-based, acyl-based, imide-based, alkyl-based, and sulfonamide-based groups. Carbamate protecting groups include tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Z or Cbz), 9-fluorenylmethyloxycarbonyl (Fmoc), 2,2,2-trichloroethoxycarbonyl (Troc). ) Group, an allyloxycarbonyl group (Alloc) group, etc., an acyl protecting group includes an acetyl (Ac) group, and an imide protecting group includes a phthaloyl (Pht) group, Examples of the alkyl protecting group include a trityl (Trt) group, and examples of the sulfonamide protecting group include a p-toluenesulfonyl group (Ts or Tos) group and a 2-nitrobenzenesulfonyl (Ns) group. The abbreviations described above are only examples, and for example, the p-toluenesulfonyl group may be described as a tosyl group with another name, such as the fat-soluble protecting group exemplified above in the present embodiment. Any group having the structure represented by the above-described group may be used.

The method for introducing the fat-soluble protecting group into the amino group of asparagine is not particularly limited, and a condensation method used for usual peptide synthesis can be used.
For example, Protective Groups in Organic Synthesis, Theodora W. et al. Greene and Peter G. M.M. Wuts, JOHN WILLY & SONS, INC. Can be used as appropriate.
For example, an amino group lipophilic protective reagent having a carboxyl group activated by an active ester such as N-hydroxysuccinimide, nitrophenol, pentafluorophenol, acetone, acetononitrile, which can dissolve the reagent, if necessary, In the presence of an organic base such as an organic amine, an inorganic base such as sodium hydrogen carbonate, a phosphate buffer, or a carbonate buffer in a mixed solvent of an organic solvent such as dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) and water. The method of making it react for 15 minutes-180 minutes under ice-cooling or room temperature is mentioned.
The amino group lipophilic protecting reagent is added in an excess amount relative to the amino group of asparagine, for example, by adding 1.1 to 3 moles of amino group liposoluble protecting reagent per 1 mol of amino group. A lipophilic protecting group can be introduced into the group.

  In the production method of the sugar chain asparagine derivative of the present embodiment, the sugar chain asparagine derivative is obtained through the alcohol precipitation step, but only in the alcohol precipitation step to which acetone, acetonitrile, diethyl ether or the like is added, if necessary. A sugar chain asparagine derivative having a purity of about 95% can be obtained. Furthermore, in order to obtain a high-purity sugar chain asparagine derivative, it can be achieved by combining desalting steps as necessary. That is, a sugar chain asparagine derivative can be obtained by adding to a silica gel resin such as ODS resin, elution with water and desalting, and then elution with an organic solvent aqueous solution. That is, by adding a sugar chain asparagine derivative and elution from the resin using an organic solvent aqueous solution of 1 to 50 times by mass with respect to silica gel resin such as ODS resin holding the sugar chain asparagine derivative, A pure sugar chain asparagine derivative can be obtained.

  Examples of the organic solvent aqueous solution include an aqueous solution of at least one organic solvent selected from the group consisting of acetonitrile, methanol, and ethanol, and the concentration of the organic solvent aqueous solution is 0.1 to 70% (v / v). And preferably 1 to 50% (v / v). In the step of eluting the sugar chain asparagine derivative with the organic solvent aqueous solution, the concentration may be gradually increased from the water and the elution may be performed, or the eluent that is the organic solvent aqueous solution may be eluted with a gradient.

  The eluted sugar chain asparagine derivative can be obtained as a powdered sugar chain asparagine derivative by concentration under reduced pressure of an organic solvent aqueous solution and drying. In the present embodiment, the obtained sugar chain asparagine derivative may be further purified by performing an alcohol precipitation step or a desalting step again.

In addition, triphenylmethyl chloride is tritylated to the amino group of asparagine in a mixed solvent of organic solvent such as DMF and DMSO and water in the presence of an organic base such as organic amine at room temperature for 4 hours to 2 days. Groups can be introduced. The purification of the sugar chain asparagine derivative can obtain a high-purity sugar chain asparagine derivative only by an alcohol precipitation step to which acetone, acetonitrile, diethyl ether or the like is added, if necessary.
As described above, in this embodiment, the alcohol precipitation step is important in the purification of the sugar chain asparagine into which the fat-soluble protecting group is introduced, and a high-purity product can be obtained only by this step without being subjected to chromatography. be able to.

  In the method for producing a sugar chain asparagine derivative, steps such as an alcohol precipitation step and a desalting step can be carried out in the same manner as described above and with reference.

  Hereinafter, the present embodiment will be described more specifically with reference to examples and comparative examples, but the present embodiment is not limited to only these examples. In addition, the measuring method used for this Embodiment is as follows.

[HPLC analysis] Using a GL Sciences HPLC GL-7400 system equipped with a glycopeptide column (Cadenza CD-C18 (Imtakt, 150 × 2 mm)), HPLC analysis was performed under the following measurement conditions.
Measurement condition:
Gradient: 2% → 17% (15 min), CH ₃ CN in 0.1% TFA solution
Flow rate: 0.3 mL / min
UV; 214 nm

[ ¹ H-NMR Measurement] 2 mg of sample was dissolved in 0.4 mL of glycopeptide D ₂ O, and ¹ H-NMR was measured using JNM-600 (600 MHz) manufactured by JEOL.

[HPLC analysis] Sugar chain asparagine (Example 1)
Using a GL Sciences HPLC GL-7400 system equipped with a column (Cadenza CD-C18 (Imtakt, 150 × 2 mm)), HPLC analysis was performed under the following measurement conditions.
Measurement condition:
Gradient: 0% → 15% (30 min), CH ₃ CN in 0.1% TFA solution
Flow rate: 0.3 mL / min
UV; 214 nm
[HPLC analysis] Sugar chain asparagine (Example 2)
Using a GL Sciences HPLC GL-7400 system equipped with a column (Mightysil RP-18 GP Aqua (Kanto Chemical, 150 × 2 mm)), HPLC analysis was performed under the following measurement conditions.
Measurement condition:
Gradient: 0% → 10% (15 min), CH ₃ CN in 0.1% TFA solution
Flow rate: 0.2 mL / min
UV; 214 nm
[HPLC analysis] Fmoc sugar chain asparagine (Example 3)
Using a GL Sciences HPLC GL-7400 system equipped with a column (Cadenza CD-C18 (Imtakt, 150 × 2 mm)), HPLC analysis was performed under the following measurement conditions.
Measurement condition:
Gradient: 20% → 100% (15 min), CH ₃ CN in 0.1% TFA solution
Flow rate: 0.2 mL / min
UV; 214 nm

LC / MS measurement was performed under the following measurement conditions. The LC and MS systems used are as follows.
[LC / MS measurement] Sugar chain asparagine (Example 1)
LC: Agilent 1100 Series Column: Imtakt, Scherzo-C18 (150 × 2 mm ID)
Column temperature: 40 ° C
Flow rate: 0.2 mL / min
UV: 214 nm
Gradient; A = water (0.05% HCOOH)
B = 0.1M HCOONH ₄ / CH ₃ CN (1/1)
B%: 2% → 100% (20min)
MS: LCQ manufactured by ThermoElectron
Ionization: ESI
Mode: Positive, Negative
[LC / MS measurement] Fmoc sugar chain asparagine (Example 3)
LC: Agilent 1100 series Column: Imtakt, Cadenza CD-C18 (150 × 2 mm ID)
Column temperature: 40 ° C
Flow rate: 0.2 mL / min
UV: 214 nm
Gradient; A = water (0.1% TFA)
B = CH ₃ CN (0.1% TFA)
B%: 20% → 100% (20 min)
MS: LCQ manufactured by ThermoElectron
Ionization: ESI
Mode: Positive, Negative

[Example 1]
Ethanol 500 mL was added to 20 chicken egg yolks and stirred well. Centrifugation was performed at 8,000 rpm for 20 minutes, and the supernatant was removed by decantation to obtain a precipitate. An operation of adding 500 mL of ethanol to the obtained precipitate, stirring well, centrifuging, and removing the supernatant was repeated three times to obtain 270 g of defatted egg yolk as a precipitate.
500 mL of water was added to 270 g of the obtained defatted egg yolk and stirred well. Centrifugation was performed at 8,000 rpm for 20 minutes, and a supernatant was obtained by decantation. The operation of adding 300 mL of water to the precipitate obtained by decantation, stirring well, centrifuging, and collecting the supernatant was repeated three times. The collected supernatant was filtered through a glass filter and concentrated under reduced pressure to 350 mL. Thereafter, the obtained concentrated solution was poured into 1500 mL of ethanol, and the resulting precipitate was centrifuged at 8,000 rpm for 20 minutes, and the supernatant was removed by decantation. The resulting precipitate was dissolved in water and poured again into ethanol. This operation was repeated three times. The precipitate produced here was collected to obtain 2.58 g of a crude purified glycopeptide.
Using 40 g of silica gel resin Wakogel (100C18) as the ODS resin, the resin was washed with methanol and then replaced with water. 2.5 g of the crude purified glycopeptide was dissolved in 20 mL of water and added to the resin after replacement with water. The resin to which the crude purified glycopeptide was added was washed with 200 mL of water, and then the glycopeptide was eluted with a 2% aqueous acetonitrile solution. The eluate was freeze-dried to obtain 212 mg of a glycopeptide.
The measurement results by HPLC and ¹ H-NMR of the obtained glycopeptide are shown in FIGS. 1 and 2, respectively. The purity by HPLC was 95%. The obtained glycopeptide was found to have a structure represented by the following formula 4 by comparison with a standard product (manufactured by Tokyo Chemical Industry) (SEQ ID NO: 2).
Formula 4:

  Furthermore, 40 g of silica gel resin Wakogel (100C18) as an ODS resin was packed in a glass column, and the resin was washed with methanol and then replaced with water. 100 mg of the glycopeptide obtained above was dissolved in 5 mL of water and added to the resin after replacement with water. After washing the resin to which the glycopeptide was added with 100 mL of water, the glycopeptide was eluted again with a 2% aqueous acetonitrile solution. The eluate was freeze-dried to obtain 70 mg of glycopeptide. The measurement result by HPLC of the obtained glycopeptide is shown in FIG. The purity by HPLC was 99%.

  10 mg of the above glycopeptide was dissolved in 0.5 mL of Tris-hydrochloric acid / calcium chloride buffer solution (50 nmM TrisHCl, 10 mM calcium chloride, 10 mM NaN3 pH 7.5) and dissolved in 0.1 mL of the same buffer. The mixture was further stirred at 37 ° C. for 3 days. Thereafter, the reaction solution was poured into 5 mL ethanol, centrifuged at 8,000 rpm for 5 minutes, and the supernatant was removed by decantation to collect the precipitate. The obtained precipitate was dissolved in 0.5 mL of water and poured into 5 mL ethanol. This operation was repeated three times. The final precipitate is dissolved in 0.5 mL of water, desalted with a water eluent by gel filtration column chromatography (PD-10 column, Sephadex G-25 resin, 8 mL), and then lyophilized to achieve the target 11 The sugar sialyl oligosaccharide asparagine 4.5 mg (yield 55.2%) was obtained.

The measurement result by HPLC of the obtained sugar chain asparagine is shown in FIG. The purity by HPLC was 95%. The obtained sugar chain asparagine was 1169.5 ([M + 2H] ²⁺ ) and 1167.7 ([M-2H] ²⁻ ) as measured by LC / MS, which was consistent with the molecular weight 2337 of the compound represented by the above formula 1. I found out that

[Example 2]
100 mg of the glycopeptide obtained in the same manner as in Example 1 was dissolved in 5 mL of Tris-HCl / calcium chloride buffer solution (50 nmM TrisHCl, 10 mM calcium chloride, 10 mM NaN3 pH 7.5) and dissolved in 1 mL of the same buffer. 50mg Kaken Pharmaceutical Actnase E was added and stirred at 37 ° C for 7 days. Thereafter, the reaction solution was poured into 20 mL ethanol, centrifuged at 8,000 rpm for 5 minutes, and the supernatant was removed by decantation to collect the precipitate. The obtained precipitate was dissolved in 1 mL of water and centrifuged at 10,000 rpm, but no precipitate was observed. In addition, sugar chain asparagine was not detected in the EtOH layer by decantation. Next, the aqueous solution was poured into 10 mL ethanol, centrifuged at 10,000 rpm for 3 minutes, and the supernatant was removed by decantation to collect the precipitate. This operation was repeated three times. The final precipitate is dissolved in 0.5 mL of water, added to gel filtration column chromatography (PD-10 column, Sephadex G-25 resin, 8 mL), and desalted by elution with water, and then freeze-dried. Crude purified 11 sugar sialyl oligosaccharide asparagine 66.4 mg was obtained. Next, column chromatography eluting with water using silica gel resin Wakogel (100C18) as ODS resin was performed to obtain 50 mg (yield 61%) of the intended 11-saccharide sialyl oligosaccharide asparagine.
The measurement result by HPLC of the obtained sugar chain asparagine is shown in FIG. As a result of LC / MS measurement, the sugar chain asparagine was 1167.7 ([M-2H] ²⁻ ), which was found to be consistent with the sugar chain asparagine represented by the above formula 1.

[Example 3]
5 mg of the sugar chain asparagine obtained in Example 2 was dissolved in 1 mL of water, 0.5 mL of DMF was added, and then 6 mg of Fmoc-OSu (N- (9-Fluorenylcarbonyl) succinimide) was dissolved in 1.5 mL of DMF. Added things. Next, 0.1 mL of 1M NaHCO ₃ was added, and the reaction was performed at room temperature for 6 hours.
Thereafter, the reaction solution was poured into 10 mL ethanol, centrifuged at 9,000 rpm for 3 minutes, and the supernatant was removed by decantation to collect a precipitate. The obtained precipitate was dissolved in 0.5 mL of water, poured into 10 mL ethanol, centrifuged at 9,000 rpm for 3 minutes, and the supernatant was removed by decantation to collect the precipitate. This operation was repeated three times. The precipitate was dissolved in 0.5 mL of water and freeze-dried to obtain 5 mg of Fmoc sugar chain asparagine in which the amino group derived from asparagine of the target sugar chain asparagine was Fmoc-converted.
The measurement result by HPLC of the obtained Fmoc sugar chain asparagine is shown in FIG. The purity by HPLC was 95%. Since the obtained Fmoc sugar chain asparagine is 1280.7 ([M + 2H] ²⁺ ) and 1278.8 ([M-2H] ²⁻ ) by LC / MS, the following formula 5 having a molecular weight of 255.89 is obtained. It was suggested that the structure is represented.
Formula 5:

  ADVANTAGE OF THE INVENTION According to this invention, the method which manufactures 11 sugar sialyl oligosaccharide asparagine simply from an avian egg defatted egg yolk on an industrial scale simply and with a sufficient yield can be provided.

SEQ ID NO: 1 is an amino acid sequence that binds to the reducing end of a sugar chain via an Asn residue in a compound represented by Formula 3.
SEQ ID NO: 2 is an amino acid sequence that binds to the reducing end of a sugar chain via an Asn residue in a compound represented by Formula 4.

Claims (11)

A method for producing an 11-saccharide sialyl-oligosaccharide asparagine represented by the following formula 1 from a defatted egg yolk:
Obtaining an 11-saccharide sialyl-oligosaccharide asparagine mixture from a defatted egg yolk, then subjecting the mixture to alcohol precipitation to obtain a sugar chain asparagine precipitate, and a desalting step of desalting the sugar chain asparagine precipitate. A method for producing an 11-saccharide sialyl-oligosaccharide asparagine.
Formula 1:
A method for producing an 11-saccharide sialyl-oligosaccharide asparagine represented by the following formula 1 from a defatted egg yolk:
An extraction process for extracting a glycopeptide extract by extracting avian egg defatted egg yolk with water or a salt solution;
A precipitation step of precipitating alcohol from the extract to obtain a glycopeptide precipitate;
Desalting the precipitate to obtain an 11-saccharide sialyl oligosaccharide peptide;
Degrading the glycopeptide with a peptide degrading enzyme to obtain an 11-saccharide sialyl-oligosaccharide asparagine mixture;
A method for producing an 11-saccharide sialyl-oligosaccharide asparagine comprising a precipitation step of precipitating alcohol from the mixture to obtain a sugar chain asparagine precipitate, and a desalting step of desalting the sugar chain asparagine precipitate.
Formula 1:
  The production method according to claim 1 or 2, wherein the desalting step of desalting the glycopeptide precipitate or the sugar chain asparagine precipitate is a step of holding the precipitate on a resin and then washing with water.   The manufacturing method of Claim 3 whose said resin is resin for reverse phase partition chromatography packing.   The production method according to claim 4, wherein the reverse phase partition chromatography packing resin is a chemically bonded silica gel resin.   The manufacturing method as described in any one of Claims 1-5 including the process of eluting with the organic-solvent aqueous solution in which the said desalting contains at least 1 sort (s) selected from the group which consists of acetonitrile, methanol, and ethanol.   The manufacturing method of Claim 6 whose density | concentration of the said organic-solvent aqueous solution is 0.1-10% (v / v). A method for producing an 11-saccharide sialyl-oligosaccharide asparagine represented by the following formula 1 from a defatted egg yolk:
Extraction process for obtaining glycopeptide extract by extracting avian egg defatted egg yolk with water,
Precipitation step of obtaining a glycopeptide precipitate by ethanol precipitation from the extract,
Desalting the precipitate with ODS resin to obtain an 11-saccharide sialyl oligosaccharide peptide;
Degrading the glycopeptide with a peptide degrading enzyme to obtain an 11-saccharide sialyl-oligosaccharide asparagine mixture;
A method for producing an 11-saccharide sialyl oligosaccharide asparagine, comprising a precipitation step of precipitating ethanol from the mixture to obtain a sugar chain asparagine precipitate, and a desalting step of desalting the sugar chain asparagine precipitate.
Formula 1:
The manufacturing method as described in any one of Claims 1-8 whose said 11 sugar sialyl oligosaccharide asparagine is a structure represented by following formula 2.
Formula 2:
A step of introducing a fat-soluble protecting group into an amino group derived from asparagine of the 11-saccharide sialyl-oligosaccharide asparagine obtained by the production method according to claim 1, and an alcohol precipitation step. A method for producing an 11-saccharide sialyl oligosaccharide asparagine into which a fat-soluble protecting group is introduced.   The lipophilic protecting group is a 9-fluorenylmethyloxycarbonyl group, benzyloxycarbonyl group, tert-butoxycarbonyl group, trityl group, 2,2,2-trichloroethoxycarbonyl group, allyloxycarbonyl group, acetyl group, The manufacturing method of Claim 10 selected from the group which consists of a phthaloyl group, p-toluenesulfonyl group, and 2-nitrobenzenesulfonyl group.