JPH11255807A - Active ester derivative of sugar-chained asparagine and synthetic intermediate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an active ester derivative of sugar-chained asparagine, by developing a derivative capable of introducing a natural sugar chain into a specific substrate through an organic chemical procedure. SOLUTION: An active ester derivative of sugar-chained asparagine is of formula I (wherein X represents formula II, in which Y is C of N; FMOC is 9-fluorenylmethyloxycarbonyl; and R<1> , R<2> , and R<3> are each H, a monosaccharide, or a sugar chain). This compd. is a derivative capable of introducing a sugar chain into an amino group-bearing derivative, and has a wide range of the applicability and a great industrial value, in that this compd. can introduce a sugar chain with cell recognition ability into a derivative of known medicines, reagents, agricultural chemicals, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an active ester derivative of an asparagine-linked oligosaccharide. Sugar chains are a group of compounds expected to be used in fields such as medicines and agricultural chemicals as their functions on cell surfaces are elucidated.

[0002]

2. Description of the Related Art The functions of sugar chains are being elucidated, but their synthesis is extremely difficult. Various methods for chemically synthesizing have been reported, but none of them has reached the level of industrially producing long-chain sugar chains.

[0003]

On the other hand, the preparation of sugar chains by enzymatic reactions has been actively studied. However, the method of successively binding monosaccharides is equivalent to a chemical method as a result. In contrast, an endoenzyme method has been reported as a method for directly introducing a natural sugar chain into a specific substrate.
For example, we have found an endo-derived from Mucor hiemalis.
By utilizing the sugar chain transfer activity of β-N-acetylglucosamine, it has been demonstrated that whole sugar chains can be transferred to a substrate having one residue of N-acetylglucosamine. However, in these methods, the amount of the enzyme supplied is limited, so it must be said that there is a difficulty in mass synthesis and the like.

[0004]

Means for Solving the Problems Accordingly, the present inventors have conducted intensive studies with the aim of developing a derivative capable of introducing a natural sugar chain to a specific substrate by an organic chemistry technique. It was found that Fmoc (9-fluorenylmethyloxycarbonyl) can be isolated from Fmoc sugar chain asparagine, and that the corresponding active ester derivative derived therefrom can easily acylate an amino compound. Reached.

[0005] That is, the gist of the present invention is to prepare an asparagine-linked sugar chain represented by the general formula 1 characterized by being produced from an asparagine derivative of the sugar chain represented by the general formula 2 in an aprotic polar solvent. An ester derivative, a synthetic intermediate thereof, and a production method.

Embedded image

Embedded image

[0006]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail.

[0007] First, the Fmoc sugar asparagine as an intermediate for the synthesis of the sugar chain asparagine active ester derivative represented by the general formula 2 will be described.

[0008] Asparagine, which is a raw material of Fmoc asparagine, has a sugar chain called a well-known asparagine-linked glycoprotein sugar chain, ie, two residues of N-acetylglucosamine, attached to a nitrogen atom of an asparagine side chain amide group.
There is no particular limitation as long as a sugar chain in which various sugar chains are bonded to the non-reducing terminal of the mother core structure consisting of three mannose residues or a sugar chain consisting of only the mother pentasaccharide is bonded. The preparation method is not limited at all, and a derivative synthesized by a chemical method may be used, but a method using a natural sugar chain is actually superior.

As a method using a natural sugar chain, a method has been reported in which a natural glycoprotein is digested with a proteolytic enzyme and isolated from a mixture of the resulting amino acid and asparagine sugar chain. For example, Tai et al. Reported a method for preparing a sugar chain asparagine from ovalbumin. (See J. Biol. Chem., Vol. 250, p. 8569, 1975.) There are no restrictions on the glycoproteins, proteolytic enzymes, or purification methods used.

[0010] As the sugar chain of asparagine sugar chain prepared as described above, a high-mannose type sugar chain, a complex type sugar chain, and a mixed type sugar chain are known, and any of them can be used. In addition, various types of sugar chains can be obtained from any type of sugar chains due to the difference in the constituent monosaccharides.
Also, a mixture may be used.

Next, the conversion of asparagine sugar chain to Fmoc will be described.

There is no limitation on the method of converting into Fmoc. Usually, asparagine-linked oligosaccharides are dissolved or suspended in a mixed solution of an aqueous base and an organic solvent, and then reacted with a well-known Fmoc agent. Well-known Fmoc agents include corresponding halides, active ester-type derivatives and the like. Specific examples include chlorides and succinimide esters, and particularly, succinimide ester derivatives (Fmoc-OS
u) is preferred.

As the base used in the aqueous base solution, a known base can be used. Examples of the inorganic base include sodium carbonate, potassium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate and the like. Examples of the organic base include tertiary amines such as pyridine and diisopropylethylamine. However, usually an inorganic base is used.

The organic solvent used is preferably a solvent miscible with water. Specific examples include methanol, ethanol, N, N-dimethylformamide, 1,4-dioxane, dimethylsulfoxide and the like. Of these, 1,4-dioxane and dimethyl sulfoxide are preferred.

In the reaction, it is possible to use an excess of the Fmoc agent, but it is usually in the range of 1.0 to 3 equivalents, preferably in the range of 1.0 to 1.5 equivalents.

The obtained Fmoc sugar chain asparagine can be easily purified by ordinary purification methods such as high performance liquid chromatography, hydrophobic chromatography, gel chromatography and ion exchange.

Next, the sugar chain asparagine active ester derivative represented by the general formula 1 which is the compound of the present invention will be described.

The compound of the present invention is an intermediate for synthesizing an active ester derivative of an asparagine-linked oligosaccharide represented by the general formula (2).
It can be prepared by deriving Fmoc sugar chain asparagine into a well-known benzotriazole-based active ester. In particular
1-hydroxybenzotriazole, 3-oxy-4-oxo-
Active esters formed from a group of compounds such as 3,4-dihydro-1,2,3-benzotriazine and a carboxylic acid can be mentioned.

Although a well-known method can be applied to the preparation, it is necessary to use a reagent having low reactivity with a sugar hydroxyl group. For example, N, N-dicyclohexylcarbodiimide or a combination thereof with N, N-dicyclohexylcarbodiimide can be used, but a so-called coupling reagent is convenient in operation.

It is important that the reaction is carried out in an aprotic polar solvent in which Fmoc sugar asparagine, which is a synthetic intermediate of the sugar chain asparagine active ester derivative represented by the general formula 2, is dissolved. As such aprotic polar solvents, specifically, N, N-dimethylformamide (DMF), N-
Examples thereof include methyl-2-pyrrolidinone (NMP), N, N, N, N, N, N-hexamethylphosphoric triamide, dimethyl sulfoxide, and formamide. DM in these
F and NMP are preferred, and NMP is particularly recommended in terms of operability and solubility.

Examples of the coupling reagent include benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP) and 2- (1H-benzotriazol-1-yl) -1,1,3,3 Well-known benzotriazole derivatives represented by the general formula 3 such as -tetramethyluronium hexafluorophosphate (HBTU).

Embedded image

As described above, when producing the compound of the present invention represented by the general formula 1, the asparagine-linked oligosaccharide derivative represented by the general formula 2 which is an intermediate thereof is dissolved in the above-mentioned solvent. It is extremely important that a solution in which these compounds are dissolved or a mixed solution with a benzotriazole derivative represented by the general formula 3 has high industrial value.

Fmoc sugar asparagine, which is a synthetic intermediate of the sugar chain asparagine active ester derivative represented by the general formula 2, which is reacted in the above aprotic polar solvent, and benzotriazol-1-yl-oxy-tris- Pyrrolidino-phosphonium hexafluorophosphate (PyBOP) and 2-
The reaction with a benzotriazole derivative represented by the general formula 3 such as (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) is not particularly limited. . Usually, 1.0 to 3.0 of the condensing reagent is used.
It can be used in the equivalent range. Preferably, 1.0 to 1.5
The range of equivalents.

Incidentally, active esters are highly reactive derivatives and are difficult to isolate and purify. Therefore, usually, a compound having an amino group is allowed to coexist during the above reaction to produce a corresponding amide derivative. The resulting amide compound having a sugar chain can be easily purified by ordinary purification methods such as high performance liquid chromatography, hydrophobic chromatography, gel chromatography, and ion exchange.

The reaction temperature of the above-mentioned reaction for producing the amide compound is not limited at all, and is in the range of 0 ° C. to the boiling point of the solvent, usually in the range of room temperature to 50 ° C. Similarly, the reaction time is not limited, and usually ranges from several hours to several days.

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited by the following Examples.

[0027]

Example 1 Ovalbumin-derived asparagine-linked oligosaccharide H-Asn (GlcNAc2Man6) -OH prepared by a method described in the literature 62.4 m
g was dissolved in 6.2 ml of a 1% aqueous sodium hydrogen carbonate solution, and a solution of 16 mg of 9-fluorenylmethyloxycarbonyloxysuccinimide (Fmoc-OSu) in 1,4-dioxane (10 ml) was added dropwise to the solution overnight. Stirred. Ether was added to the reaction solution, the layers were separated, and the aqueous layer was washed with ether. PH of aqueous layer is 3-4
Was adjusted with citric acid. This aqueous solution is applied to Amberlite XA
The mixture was passed through the column of D-2, and sufficiently washed with water. Subsequently, it was eluted with methanol and concentrated under reduced pressure. When dissolved in water again and freeze-dried, 63 mg of Fmoc-Asn (GlcNAc2Man6) -OH was obtained as a white powder. The structure was confirmed by MALDI-TOFMS.
Observed 2870, Calculated [M + Na] = 2871.

Embedded image

[0028]

Example 2 Asparagine-linked oligosaccharides derived from ovalbumin were converted to Fmoc in the same manner as in Example 1 while maintaining the mixture of the sugar chains. H-
When Asn (sugar chain) -OH 101 mg was used and the sugar chain structure was assumed to be the sugar chain structure used in Example 1, 92.7 mg of Fmoc
-Asn (sugar chain) -OH was obtained. High Performance Liquid Chromatography (Inertsil
ODS3; 0.1% trifluoroacetic acid-acetonitrile (20-30% /
30 min); 1 ml / min) to separate and isolate each component, and the MALDI-TOF
MS analysis revealed that the mixture had the following sugar chains. Man4GlcNAc1-Man3GlcNAc2-; found 1919, calculated [M + Na] = 1918, Man4-Man3GlcNAc2-; found
2119, Calculated [M + Na] = 2121, Man2GlcNAc2-Man3GlcNAc2-;
Actual value 2021, calculated value [M + K] = 2016, Man2GlcNAc3-Man3GlcNA
c2-; actual measured value 2189, calculated value [M] = 2197, Man2GlcNAc2-Man3Glc
NAc2-; observed 1985, calculated [M] = 1977, Man3-Man3GlcNAc2
-; Actual value 1736, calculated value [M] = 1733, Man5-Man3GlcNAc2-; actual value 2063, calculated value [M] = 2057, Man2-Man3GlcNAc2-; actual value 1
572, calculated value [M] = 1571, GlcNAc3-Man3GlcNAc2-; found 185
8, calculated value [M] = 1856. Assuming that all sugar chains were the sugar chains of Example 1, the yield was 81%.

[0029]

Example 3 Fmoc-Asn (GlcNAc2Man) prepared in Example 1
6) 5.0 mg of -OH was dissolved in 2 ml of NMP. To this was added 2.9 μl of a 1 M diisopropylethylamine / NMP solution at room temperature and 1.5 mg of benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP),
Then, 3.3 mg of 6-mono-amino-β-cyclodextrin was added as an amino compound for capturing the generated active ester, and the mixture was stirred overnight. Tested by the same high performance liquid chromatography as in Example 2, a cyclodextrin derivative having a natural sugar chain on the side chain where the active ester derivative was reacted with an amino group was obtained in a yield of 74%. When a similar reaction was performed using 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole, the yield was Was 54%.

[0030]

Example 4 Fm having various sugar chains prepared in Example 2
10 mg of a mixture of oc-Asn (sugar chain) -OH was dissolved in 1 ml of NMP, and benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBOP) was dissolved in the presence of molecular sieves 4A in the same manner as in Example 3. ) 2.8mg
To form an active ester, and one equivalent of alanine methyl ester was coexisted as an amino compound for capturing the active ester. 83% as determined by high performance liquid chromatography
Fmoc-Asn (sugar chain) -Ala-OMe was obtained in a yield of

[0031]

As described above, the compound of the present invention is a derivative that can introduce a sugar chain into a derivative having an amino group, and can introduce a sugar chain having cell recognition ability into a derivative of a known drug, reagent, or pesticide. In this respect, its application range is very wide, and its industrial value is great.

Claims (8)

[Claims] 1. A compound of the general formula A sugar chain asparagine active ester derivative represented by the formula: 2. A general formula obtained by 9-fluorenylmethyloxycarbonylation of asparagine-linked asparagine obtained by treating a glycoprotein with a protease. The synthetic intermediate of the sugar chain asparagine active ester derivative represented by 3. A sugar chain asparagine active ester derivative according to claim 1, which is dissolved in an aprotic polar solvent capable of dissolving a synthetic intermediate of the sugar chain asparagine active ester derivative represented by the general formula. 4. The synthetic intermediate of an asparagine active ester derivative of a sugar chain according to claim 2, wherein the sugar chain is a high mannose type sugar chain. 5. Synthesis of an asparagine-active ester derivative of a sugar chain represented by the general formula (2), wherein the intermediate is dissolved in an aprotic polar solvent capable of dissolving an intermediate. Intermediate. 6. An aprotic polar solvent capable of dissolving a synthetic intermediate of a sugar chain asparagine active ester derivative represented by the general formula:
General formula A composition for synthesizing a sugar chain asparagine active ester derivative comprising a benzotriazole derivative represented by the following formula and a synthetic intermediate of the sugar chain asparagine active ester derivative according to claim 2. 7. N-methyl-2-pyrrolidinone or N, N-dimethylformamide is used as an aprotic polar solvent capable of dissolving an intermediate for synthesizing a sugar chain asparagine active ester derivative represented by the general formula: The composition for synthesizing an active sugar ester asparagine derivative according to claim 6, wherein the composition is used. 8. An aprotic polar solvent which can dissolve a synthetic intermediate of an asparagine active ester derivative of a sugar chain represented by the general formula:
3. A process for producing an active sugar chain asparagine derivative, comprising reacting a benzotriazole derivative represented by the following general formula with a synthetic intermediate of an active sugar ester asparagine derivative represented by claim 2.