JP2000319297A - Preparation of enzymatically stable complex glycopeptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject enzymatically stable complex glycopeptide that can be expected to improve the stability and pharmaceutical action in situ and is useful for medicines by preparing a complex glycopeptide that is stable to an enzyme peptide: N-glycanase. SOLUTION: The objective compound is obtained by preparing a complex glycopeptide derivative that is stable to an enzyme peptide: N-glycanase (EC. 3. 5. 1. 52) preferably bearing a saccharide chain linking to the residual group of L-glutamine(Gln) in the peptide via the N-glycoside bond. The saccharide chain is introduced by using 9-fluorenylmethyloxycarbonyl-glutaminyl-N- acetylglucosaminide prepared by linking N-acetyl-D-glucosamine (GlcNAc) to Gin as a synthetic unit, to synthesize the glycopeptide to which GlcNAc is linked at the Gln residue through the solid phase method and by transfer-adding a saccharide chain to the resultant glycopeptide by using an enzyme: endo-β-N- acetylglucosaminidase.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for preparing a glycoconjugate which is stable to a sugar chain hydrolase. More specifically, the enzyme peptide N-glycanase (EC. 3.5.1.
52) The present invention relates to a method for preparing a glycoconjugate derivative which is stable against 52). The present invention is applied to medicine.

[0002]

2. Description of the Related Art A complex glycopeptide or complex glycoprotein comprises a peptide or protein body and a sugar chain portion.

[0003] The sugar chain of a complex glycopeptide or complex glycoprotein is composed of an N-linked sugar chain having an N-glycosidic bond to L-asparagine (Asn) and an O-glycan linked to L-serine (Ser) or L-threonine (Thr). There are O-linked sugar chains linked by glycosides.

[0004] The N-linked sugar chain is a peptide moiety in which the hydroxyl group at position 1 of GlcNAc at the reducing end of the sugar chain having a core structure consisting of three D-mannose (Man) and two N-acetyl-D-glucosamine (GlcNAc) is a peptide moiety. Has an N-glycosidic bond in β-coordination with β-carboxamide of Asn.

[0005] The sugar chain is a high mannose type sugar chain in which several more Mans are linked to Man of the core structure, GlcNAc, D-galactose (Ga
l), complex sugar chains in which a plurality of types of sugars such as sialic acid (NeuAc) are bonded, and hybrid sugar chains in which both types are mixed.

[0006] These complex glycopeptides or glycoproteins are attacked by various hydrolytic enzymes in the living body as well as the peptide or protein itself, and are degraded and digested.

[0007] As the N-linked sugar chain hydrolase, exo (ex.
o) In addition to the type of glycolytic enzyme, GlcNAcβ1
→ There is an endo-β-N-acetylglucosaminidase that cuts the 4GlcNAc bond and a peptide that hydrolyzes the amide bond between the reducing terminal sugar GlcNAc and Asn: N-glycanase.

[0008] Peptide: N-glycanase (Peptide: N-gl
Ycanase) is the official name Peptide -N ⁴ - (N-acetyl-beta-D-
Glucosaminyl) asparagine amidase, enzyme number EC
3.5.1.52, usually called glycopeptidase, N-glycanase or PNGase.

Peptides: N-glycanase is described in Takahashi et al. [Biochem. Biophys. Res. Commun., Vol. 76, No. 1194.
Page (1977)] obtained from almond seeds
N-glycopeptidase A (PNGase A), TH pramer Jr.
(THPlummer Jr.) et al. [Journal of Biological Chemistry (J. Biol. Chem.) Vol.
00 (1984)], a bacterium found by Flavobacterium meningosepticum
Glycopeptidase F (PNGase F) is recently known, and recently Suzuki et al. [Proc. Natl. Acad. S.
ci., USA), Vol. 92, p. 6244 (1997)] have discovered a new enzyme (PNGase HO) in the oviducts of chickens.

[0010]

The sugar chains of complex glycopeptides or complex glycoproteins play important roles in cell-cell recognition, substrate-receptor mutual recognition, or stability and metabolism.

[0011] However, in vivo, a change in function is caused by the digestive action of the hydrolase. In particular, when the peptide is attacked by N-glycanase, the sugar chain is cut off from the base, the function of recognizing the sugar chain and maintaining the structure is lost, and the peptide or protein itself becomes unstable to metabolism.

[0012] In order to maintain the function of the sugar chain of the complex glycopeptide or complex glycoprotein, there is a need for the development of a method for stabilizing peptide: N-glycanase.

[0013]

Means for Solving the Problems Peptide: The following two methods are considered as methods for stabilizing the sugar chain of a complex glycopeptide or complex glycoprotein against N-glycanase attack. That is, 1) a complex glycopeptide or complex glycoprotein derivative having a structure stable to an enzyme is synthesized. 2) Develop derivatives that inactivate enzymes or act as inhibitors.

The biologically N-linked sugar chain is attached to Asn-X-
Ser / Thr (X is any amino acid except proline, Ser / Thr
Is either Asn of the recognition sequence represented by Ser or Thr), and attaches a sugar chain biologically to Asn without this recognition sequence or a Gln residue that is one methylene chain longer than Asn. Asn without Gln residue or recognition sequence
A complex glycopeptide or glycoprotein having a sugar chain attached to a residue does not exist at all in nature.

As a method for synthesizing an unnatural glycoconjugate, the present inventors have previously described a method in which a G-linked Asn is linked to an N-glycoside.
A method for synthesizing a glycopeptide derivative chemically and enzymatically by synthesizing a glycopeptide derivative containing lcNAc and transferring a naturally-occurring sugar chain thereto with the enzyme endo-β-N-acetylglucosaminidase (Endo enzyme) [Carbohydrate Hydrate Research (Carbohydr. Res.) Vol. 292, p. 61 (1996)]. By this method, it became possible to add a sugar chain to any Asn of the peptide without a recognition sequence.

Instead of using a synthetic unit in which GlcNAc is bound to Asn, 9-fluorenylmethyloxycarbonyl (Fmoc) -glutaminyl-N-acetylglucosaminide [Fmoc- is obtained by binding GlcNAc to L-glutamine (Gln). Gln (GlcN
Ac) -OH] as a synthesis unit, and GlcNAc
Is synthesized by a solid phase method, and
do enzyme eg Endo enzyme from Mucor hiemalis (Endo-M)
It has become possible to synthesize a novel complex glycopeptide having a sugar chain at a Gln residue, which is not biologically possible, by transferring a sugar chain. For example, the method of the present inventors [JP-A-9-31095] may be followed.

In the present invention, Gln of a peptide is
Introduce a sugar chain to the residue. As the sugar chain, any of a high-mannose type, a complex type, and a mixed type sugar chain can be added.

Peptide: N-glycanase hydrolyzes the amide bond between GlcNAc and Asn by recognizing several amino acids of the recognition sequence together with the sugar chain portion of the glycopeptide or glycoprotein.

An enzyme peptide that hydrolyzes an amide bond between a sugar chain and Asn: N-glycanase is a complex glycopeptide having a sugar chain attached to Gln or a Asn having no recognition sequence, which is longer by one methylene chain than Asn. Did not predict at all.

The present invention provides a novel glycopeptide derivative in which a sugar chain is amide-linked to a Gln residue, which is stable without being affected by peptide: N-glycanase. Is found to have an inhibitory effect on

That is, the present invention provides a method for preparing a complex glycopeptide derivative in which a sugar chain is added to the Gln residue of a peptide which is a peptide and which is stable and an inhibitor against N-glycanase.

The concept of the present invention is based on the difference in the ability to recognize the amino acid portion of the peptide of peptide: N-glycanase, and is applicable to complex glycopeptides and proteins in which a sugar chain is added to Asn without a recognition sequence. .

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION The following two forms are assumed for a derivative having a sugar chain at the Gln residue of a peptide. One is a derivative in which Asn, which originally has a sugar chain, is replaced with Gln.
One is a derivative obtained by adding a sugar chain to Gln, which originally had no sugar chain.

These peptide derivatives in which a sugar chain has been added to the Gln residue can be used as peptide: N-glycanase-stable physiologically active complex glycopeptide, or by inhibiting the enzyme activity to form the original complex glycopeptide or complex glycoprotein. It is used in medicine and medical treatment as a stabilizing drug.

[0025]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Here, three cases which are the basis of the present invention will be described. That is, 1) a method for synthesizing a complex glycopeptide having a sugar chain at a Gln residue. 2) The amide bond of the sugar chain of the synthetic glycopeptide to Gln is stable to peptide: N-glycanase. 3) The synthetic glycoconjugate serves as an enzyme inhibitor of peptide: N-glycanase. However, the invention is not limited to this embodiment.

[0026]

Example 1 Preparation of complex glycopeptide in which Asn residue of peptide having N-linked sugar chain was substituted with Gln: Glycopeptide SGP (1) having a sugar chain having exactly the same sugar chain structure as human transferrin was prepared. From chicken egg yolk to Sekoura [Biochem. Biophys. Acta] Vol. 1335,
23 (1997)]. SGP: H-Lys-Val-Ala-Asn (CHO) -Lys-Thr-OH (1) Here, CHO is a sialo double-stranded complex sugar composed of (NeuAc-Gal-GlcNAc-Man) ₂ -GlcNAc ₂ Indicates a chain.

The peptide portion of SGP is converted to a protease (prona
se), followed by gel filtration and purification on Sephadex G-25 to give a sugar amino acid S in which the peptide moiety is only Asn.
GN (2) was prepared. SGN: H-Asn (CHO) -OH (2)

Glycopeptide QSGP in which Asn of SGP is substituted with Gln
(3) was synthesized by the following procedures 1) and 2). QSGP: H-Lys-Val-Ala-Gln (CHO) -Lys-Thr-OH (3) 1) Using Fmoc-Gln (GlcNAc) -OH as a synthesis block,
Developing solid phase synthesis of peptides by the moc method,
Glycopeptide QGP (4) having cNAc was synthesized. QGP: H-Lys-Val-Ala-Gln (GlcNAc) -Lys-Thr-OH (4)

2) This glycopeptide QGP (4) is used as a receptor and S
Using GN (2) as a sugar chain donor, a novel complex glycopeptide derivative QSGP (3) having a sialo complex-type sugar chain at a Gln residue was synthesized by a sugar chain transfer reaction using an enzyme Endo-M.

[0030]

Example 2 Synthesis of a complex glycopeptide derivative in which a sugar chain is introduced into the Gln residue of a physiologically active peptide having no sugar chain: a neuropeptide involved in pain transmission and intestinal (ileal) contraction consisting of 11 amino acids N- for substance P (SP, 5)
There are two Gln residues at positions 5 and 6 from the end. SP: H-Lys-Pro-Arg-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-
NH ₂ (5)

The glycopeptide derivative having GlcNAc introduced into Gln at the 5- or 6-position of SP was prepared according to the method of the present inventors [Japanese Patent Application No. 9-34].
3979]. Then, SGN (2) was used as a sugar chain donor, and the sugar chain transfer reaction by Endo-M was performed to obtain the 5- or 6-position.
Derivative SP5-STF (6) with a sugar chain added to Gln at position
SP6-STF (7) was synthesized. SP5-STF: H-Lys-Pro-Arg-Pro-Gln (CHO) -Gln-Phe-Phe-Gl
y-Leu-Met-NH ₂ (6) SP6-STF: H-Lys-Pro-Arg-Pro-Gln-Gln (CHO) -Phe-Phe-Gl
y-Leu-Met-NH ₂ (7)

[0032]

Example 3 Effect of Peptide: N-Glycanase on SGP and QSGP: Using SGP and QSGP as substrates, two peptides of bacterial PNGase F and almond-derived PNGase A: N-glycanase were reacted. The two enzymes were reacted under the following conditions using Boehringer Mannheim reagents. Substrate (SGP or QSGP) 0.5 mM, buffer 50
mM (pH 7.8 phosphate buffer for PNGase F, pH 5.0 acetate buffer for PNGase A), enzyme (50 U / ml for PNGase F, P
(12.5 mU / ml for NGase A) at 37 ° C for 2 to 6 hours.
For detection and quantification of substrates and degradation products, a reversed-phase (C18) column was used, and a water-acetonitrile mixed solvent containing 0.1% trifluoroacetic acid (TFA) was used as a developing solution. By chromatography (HPLC). Table 1 shows the results.

[0033]

[Table 1]

The sugar chains of SGP bound to Asn are PNGase F and
The peptide (H-Lys-Val-Ala-Asp-Lys-Thr-OH), which is rapidly hydrolyzed by any of the enzymes in A to convert Asn with a sugar chain to L-aspartic acid (Asp) Sugar chains were formed. However, QSGP in which Asn was replaced by Gln was stable without any degradation by any enzyme.

[0035]

Example 4 Effect of peptide: N-glycanase on substance P (SP) sugar chain derivative: SP derivative SP5-STF having a sugar chain at Gln at position 5 or 6 prepared in Example 2
The effect of PNGase F on (6) and SP6-STF (7) was examined. The reaction was carried out according to Example 3 using 50 μM as the substrate. The reaction was performed for 3 hours. SGP was used as a control. Table 2 shows the results.

[0036]

[Table 2]

The sugar chains of the derivatives obtained by adding a sugar chain to Gln at the 5th and 6th positions of SP did not undergo any hydrolysis by PNGase F, and these SP derivatives were stable.

[0038]

Example 5 Inhibitory Effect of QSGP on Hydrolysis by SGP Peptide: N-Glycanase: QSGP on SGP Hydrolysis by Peptide: N-Glycanase When QSGP Coexists in Enzyme Reaction System Using SGP as Substrate The effect of was investigated. SGP 0.25 mM, QSGP 0-0.25 mM, PNGase F 25 U / ml,
The reaction time was 2 hours, and the other conditions were the same as in Example 3. Table 3 shows the results.

[0039]

[Table 3]

SGP alone hydrolyzed 98% in 2 hours of reaction, but in the presence of QSGP, hydrolysis of SGP peptide: N-glycanase was inhibited, and the degradation rate of SGP was reduced.

[0041]

EFFECT OF THE INVENTION The present invention provides a glycoconjugate that is enzymatically stable in vivo and is effective as an inhibitor of peptide: N-glycanase. It is expected that the stability and pharmacological action of the physiologically active glycopeptide in the body will be improved.

Claims (3)

[Claims] 1. An enzyme peptide: N-glycanase (EC. 3.
5. A method for preparing glycoconjugate derivatives that are stable against 52). 2. The method according to claim 2, wherein the L-glutamine (Gln) residue of the peptide is N-
2. The method according to claim 1, which is a complex glycopeptide derivative having a glycoside-linked sugar chain. 3. An enzymatic peptide prepared by the method of claim 1 or 2, which is a complex glycopeptide derivative that is stable to N-glycanase.