JP2000302800A - Sugar chain calcitonin derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new compound useful as a medicine by replacing 26-position aspartic acid to an N-bond type sugar chain-containing asparagine residue. SOLUTION: This calcitonin derivative is obtained by replacing 26-position aspartic acid to an N-bond type sugar chain-containing asparagine residue. The derivative is preferably an eel calcitonin derivative, more preferably an eel calcitonin derivative obtained by replacing an 26-position aspartic acid of the formula I [R is (NeuAc-Gal-GlcNAc-Man)2-Man-G1cNAc, (Gal-GlcNac- Man)2-Man-GlcNac or Man6-GlcNac (NeuAc is sialic acid); Gal is D-galactose; GlcNAc is N-acetyl-D-glucosamine; and Man is D-mannose] to an N-bond type sugar chain-containing asparagine residue. The derivative can be synthesized by subjecting a saccharide-eel calcitonin derivative of formula II to sugar chain transfer reaction of enzyme endoglycosidase.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel calcitonin derivative having a sugar chain and a method for producing the same. The present invention is applied to medicine.

[0002]

2. Description of the Related Art Peptide hormone calcitonin (abbreviated as CT) consisting of 32 amino acids functions as a calcium-regulating hormone in mammals and suppresses bone resorption, so that calcitonin in humans, pigs, salmon, eels, etc. Alternatively, a derivative thereof has been used as a therapeutic agent for osteoporosis and the like.

[0003] When a physiologically active peptide is used as a drug, its effectiveness is often significantly impaired due to decomposition by a decomposing enzyme in a body fluid, and it is sometimes absorbed halfway and does not reach a target organ. Overcoming the points is an important issue.

[0004] Glycoconjugates exist in cells and body fluids in the form of glycoproteins and glycolipids, and sugar chains are deeply involved in cell substrate recognition and cell-cell recognition. In addition, sugar chains are involved in the absorption and decomposition and stability of physiologically active peptides and proteins in vivo.

[0005] Among physiologically active peptides such as peptide hormones, those having sugar chains such as human chorionic stimulating hormone hCG are known, and play a role in regulation and stability of physiological functions.

On the other hand, adding a sugar chain to a physiologically active peptide having no sugar chain such as calcitonin is expected to be useful for improving physiological functions and enhancing stability.

[0007] It is not biologically possible to add a sugar chain to a physiologically active peptide originally having no sugar chain. However, Haneda et al. [Carbohydrate Research (Carb
Res.), Vol. 292, pp. 61-70 (1996)] previously described a method for the chemical-enzymatic synthesis of complex glycopeptides based on the glycosylation of glycopeptides and the transglycosylation reaction of the enzyme endoglycosidase. reported.

[0008] The L-asparagine (Asn) residue of the peptide has N
Glycopeptides to which -acetyl-D-glucosamine (GlcNAc) has been added are described in, for example, Inazu et al. [Peptide Chemistry 1995
(Peptide Chemistry 1995), pp. 61-64 (1996)], and synthesized by a solid phase method without protection of sugar hydroxyl groups. On the other hand, a natural sugar chain is added to the GlcNAc residue of this glycopeptide by a sugar chain transfer reaction of, for example, an endo-β-N-acetylglucosaminidase (endo-M) enzyme derived from Mucor hemisalis to form a complex glycopeptide. Can be synthesized.

By using this technique, Mizuno et al. [Tetrahedron Lett., Et al.] Added a 3 position Asn residue of calcitonin having no sugar chain such as eel calcitonin.
39, pp. 55-58 (1998)] introduces GlcNAc, followed by Haneda et al. [Bioorganic Medicinal Chemistry Letter (Bioorg. Med. Chem. Lett.), Vol.
1303-1306 (1998)] synthesized an eel calcitonin derivative into which an N-linked sugar chain was introduced.

[0010] It has been recognized that eel calcitonin to which these sugars and sugar chains are added has a hormone (calcium metabolism regulation) activity in a living body that is equal to or stronger than calcitonin itself.

[0011]

SUMMARY OF THE INVENTION The eel calcitonin
The amino acid at position 26 is L-aspartic acid (Asp). However, since the amino acid at position 26 of salmon calcitonin is Asn, the amino acid at position 26 of eel calcitonin is changed to Asn.
It is expected that there will be no significant change in activity even if it is changed to.

A derivative in which Asp at position 26 was replaced by Asn was synthesized by a conventional solid phase synthesis method of a peptide, and it was found that there was no significant change in hormonal activity even when Asp at position 26 in eel calcitonin was replaced with Asn.

By replacing Asp at position 26 with Asn, sugars and sugar chains can be introduced into the Asn residue at position 26 using the above-described chemo-enzymatic method. The calcitonin derivative into which these sugars and sugar chains are introduced is expected to have enhanced activity and change in pharmacokinetics.

An object of the present invention is to provide a novel sugar chain-calcitonin derivative in which the Asp residue at position 26 of calcitonin is substituted by an Asn residue having an N-linked sugar chain.

[0015]

SUMMARY OF THE INVENTION A novel calcitonin derivative in which the Asp residue at position 26 is substituted with an Asn residue having an N-linked sugar chain, for example, an eel calcitonin derivative shown in (Chemical Formula 1), A sugar-eel calcitonin derivative represented by the formula (Formula 2) in which the group is substituted with an N-acetyl-D-glucosaminyl asparagine [Asn (GlcNAc)] residue is converted into a naturally occurring N by a sugar chain transfer reaction of an enzyme endoglycosidase. -It is synthesized by adding a linked sugar chain.

Embedded image

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The synthesis of a derivative of eel calcitonin obtained by substituting aspartic acid at position 26 with Asn (GlcNAc) may be carried out by any method. For example, Inazu et al.
598 (1998)] found that GlcNAc was added to Asn at position 3 of eel calcitonin.
Can be used according to the method used for the synthesis of the derivative into which is introduced. Toshima et al. [Peptide chemistry 1
996 (Peptide Chemistry 1996), pp. 25-28 (1997)]
It can also be synthesized according to the method developed by Suzuki.

Using the glycopeptide having a GlcNAc residue as an acceptor, a sugar chain transfer addition reaction with an endoglycosidase from a sugar chain donor is performed in the following procedure.

The endoglycosidase used in the present invention includes endo-β-N-acetylglucosaminidase (EC
3.2.1.96), for example, Mucourt Himaris (Muco
r hiemalis) is used. The enzyme is a sugar represented by R cut between acetylchitobiose (GlcNAc-GlcNAc) of a sugar chain donor represented by [R-GlcNAc-Asn] prepared by treating a natural glycoprotein with a protease. The chain portion is transferred to the GlcNAc portion using, for example, a sugar derivative of eel calcitonin represented by (Chem. 2) as an acceptor, and a calcitonin derivative having a sugar chain represented by (Chem. 1) is synthesized.

As the sugar chain donor represented by [R-GlcNAc-Asn], a complex type sugar chain, a high mannose type sugar chain, or a mixed type sugar chain is used, so that the corresponding target compound (Chem. 1) ) Is obtained.

As a complex type sugar chain, for example, when R is (NeuAc-
The complex type sugar chain having sialic acid which is (Gal-GlcNAc-Man) ₂ -Man-GlcNAc is prepared from human transferrin or egg yolk. When sialic acid is removed by the enzyme sialidase, R
(Gal-GlcNAc-Man) ₂ -Man-GlcNAc is prepared asialo complex type sugar chain. A high-mannose type sugar chain consisting of _six mannoses whose R is represented by Man ₆ -GlcNAc is prepared from ovalbumin. Enzymatically or chemically modified sugar chains or chemically synthesized sugar chains can also be used.

The reaction of the present invention is carried out by mixing a sugar chain donor, a sugar calcitonin derivative which is a sugar chain acceptor, and an enzyme endoglycosidase in a buffer. An example of the composition of the reaction solution is as follows: sugar chain donor 25 mM, sugar chain acceptor
10 mM, enzyme endo-M, 30 mU / ml, pH 6.25, 60 mM phosphate buffer, and the reaction is performed at 37 ° C. for 1 to 2 hours.

The reaction product in the enzyme reaction solution was analyzed using a C18 reverse phase system (ODS) column and 0.1% trifluoroacetic acid (TDS).
This is carried out by high-performance liquid chromatography (HPLC) using a water-acetonitrile solution containing FA) as a developing solvent by measuring the ultraviolet terminal absorption at 210 nm.

The sugar chain calcitonin produced is usually ODS
It is isolated by lyophilization after separation by HPLC using a column.

[0024]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

[0025]

Example 1 Synthesis of an eel calcitonin derivative in which Asp at position 26 was substituted with an Asn residue having a sialo complex type sugar chain: (1) Asp (position 2) was substituted with Asn (GlcNAc) residue Preparation of eel calcitonin derivative [Asn (GlcNAc) ²⁶ ] -CT: Toshima et al. [Peptide Chemistry 1996 (Peptide C
hemistry 1996), pp. 25-28 (1997)]
It was synthesized by the solid phase Boc strategy. HPLC [ODS
(YMC Pak ODS-AM, φ4.6x150mm), developing solvent 0.1% trifluoroacetic acid (TFA) containing 20-70% acetonitrile (0-40
) At a flow rate of 1.2 ml / min], and the target substance was detected as a peak with a retention time of 14.3 minutes. In MALDI-TOFMS analysis, a main ion peak was observed at m / z [M + H] ⁺ 3617.4, and [Asn (GlcNAc) ²⁶ ]-
CT (molecular formula C ₁₅₄ H ₂₅₅ O ₅₁ N ₄₅ S ₂ , molecular weight 3617.1) was confirmed.

(2) Preparation of Sugar Chain Donor: Egg yolk to Seki et al. [Biochimica Bacteria Acta (Biochim. B)
Acta), Vol. 1335, pp. 23-32 (1997)], treating the glycopeptide SGP having a sialo complex type sugar chain with pronase, and then filtering the peptide portion with Asn residues by Sephadex G-25 gel filtration. The only sialo complex type sugar amino acid SGN [(NeuAc-Gal-GlcNAc-Man) ₂ -Man-GlcNAc ₂ -Asn
(Molecular weight 2338)].

(3) Sugar chain transfer reaction: sialo-complex type sugar amino acid SGN 25 mM, [Asn (GlcNAc) ²⁶ ] -CT 10 m shown in Chemical formula 2
M, 60 mM pH6.25 phosphate buffer, endopurified from the culture of Mucor hiemalis by the method of Kadowaki et al. [Journal of Biochemistry (J. Biochem.), Vol. 110, pp. 17-21 (1991)] Reaction solution 40 containing -m enzyme 30 mU / ml
μl was reacted at 37 ° C. for 1 hour. After the reaction was stopped by adding an equal volume of cold 1% TFA solution, the reaction product was subjected to HPLC [ODS (Mig
htysil RP-18, φ6 x 250mm, Kanto Chemical), developing solvent 0.1%
30-35% acetonitrile containing TFA (0-40 minutes), flow rate 1.2m
l / min] at 210 nm. Glycosyltransfer reaction product from amino acid SGN such as sialo complex type to sugar chain receptor [Asn (GlcNAc) ²⁶ ] -CT has a retention time of HPLC analysis of 30.1 minutes (glycan receptor [Asn (GlcNAc) ²⁶ ] -CT , 37.1 min), and the reaction yield (based on sugar chain acceptor, molar yield) was 15.0%.

(4) Isolation and identification of reaction product: A peak substance in HPLC analysis corresponding to a sugar chain transfer reaction product was isolated by HPLC fractionation. MALDI TOF-MS analysis result, m / z [MH] ^-
A main ion peak was recognized at 5619.8, and the main ion peak corresponded to the asialo form when further treated with sialidase.
Move to m / z value, ((NeuAc-Gal-GlcNAc-Man) ₂ -Man-GlcNA
calcitonin derivative _{[C 230 H 378 O 1 07} N 50 S 2 having made of c _2] disialo double stranded complex type sugar chain, it was confirmed that the molecular weight 5619.9.

[0029]

Example 2 Asn having an asialo complex type sugar chain at Asp 26
Synthesis of Eel Calcitonin Derivative Substituted at Residue: Asialoglycosaccharide Amino Acid Prepared by Further Treating Egg Yolk-Derived Sialoglycosaccharide SGN Prepared in Example 1 with Sialidase and Sephadex G-25 Gel Filtration as Sugar Chain Donor AS
GN [(Gal-GlcNAc-Man) ₂ -Man-GlcNAc ₂ -Asn (molecular weight 175
6)], and reacted under the same reaction conditions as in Example 1.
Under the same HPLC analysis conditions, asialo complex type sugar amino acid ASGN
Glycosyltransfer reaction product from the glycan receptor [Asn (GlcNAc) ²⁶ ] -CT to the glycan receptor [Asn (GlcNAc) ²⁶ ] -CT
lcNAc) ²⁶ ] -CT, 37.1 min), and its reaction yield (to sugar chain acceptor, molar yield) was 11.2%. The peak substance of HPLC analysis corresponding to the transglycosylation reaction product was isolated by HPLC fractionation, and as a result of MALDI TOF-MS analysis,
A main ion peak was observed at m / z [M + H] ⁺ 5035.7, and [(Gal
_{-GlcNAc-Man) 2 -Man-GlcNAc} 2] calcitonin derivative having asialo complex type sugar chain consisting of _{[C 208 H 344 O 91 N} 48 S 2, it was confirmed that the molecular weight 5037.4.

Asp having a high mannose type sugar chain at Asp at position 26
Synthesis of Eel Calcitonin Derivatives Substituted with Residues: As a sugar chain donor, ovalbumin is treated with pronase, sephadex G-25 gel filtration, and further purified by Dowex 50 ion exchange chromatography. The reaction was carried out using the amino acid M ₆ GN [Man ₆ -GlcNac ₂ -Asn (molecular weight: 1511)] under the same reaction conditions as in Example 1. Under the same HPLC analysis conditions, HPLC analysis
Sugar chain receptor from high-mannose type sugar amino acid M ₆ GN [Asn (Glc
The transglycosylation reaction product to NAc) ²⁶ ] -CT was detected as a peak at a retention time of 31.8 minutes in HPLC analysis, and the reaction yield (vs. sugar chain receptor, molar yield) was 3.9%. The peak substance of the HPLC analysis corresponding to the transglycosylation reaction product was isolated by HPLC fractionation, and as a result of MALDI TOF-MS analysis, m / z [M + H] +
A main ion peak was observed at 5.0, and a calcitonin derivative [C6-Clc having a high mannose-type sugar chain consisting of [Man6-GlcNAc2]
_{19 8 H 328 O 86 N 46} S 2, it was confirmed that the molecular weight 4793.2.

[0031]

EFFECT OF THE INVENTION By converting a constituent amino acid of the physiologically active peptide calcitonin into an amino acid capable of introducing a sugar chain and synthesizing a derivative having a sugar chain introduced therein, the stability in blood or the body is improved and the pharmacological action is improved. It is possible to provide a highly effective calcitonin derivative, which is effective when used as a medicine.

Claims (4)

[Claims] 1. A calcitonin derivative in which aspartic acid at position 26 is substituted with an asparagine residue having an N-linked sugar chain. 2. An eel calcitonin derivative in which aspartic acid at position 26 is substituted with an asparagine residue having an N-linked sugar chain. 3. An eel calcitonin derivative in which aspartic acid at position 26 represented by the following formula (Formula 1) is substituted by an asparagine residue having an N-linked sugar chain. Embedded image In the formula, R is (NeuAc-Gal-GlcNAc-Man) ₂ -Man-GlcNAc, (Gal-
GlcNAc-Man) ₂ -Man-GlcNAc or Man ₆ -GlcNAc (NeuAc is sialic acid, Gal is D-galactose, GlcNAc is N-
Acetyl-D-glucosamine, Man indicates D-mannose). 4. The method according to claim 4, wherein the sugar chain of the complex saccharide is transferred to a calcitonin derivative in which aspartic acid at position 26 is substituted with an N-acetyl-D-glucosaminyl asparagine residue in the presence of endoglycosidase. A method for producing a calcitonin derivative having an N-linked sugar chain added to a group.