JP2916297B2 - Phosphopeptide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel phosphopeptide.

[0002]

2. Description of the Related Art In recent years, PHF (paired helical filament) has been added to the brain of an aged person, particularly the brain of a patient with Alzheimer's dementia.
s), and it has been revealed that highly phosphorylated tau (τ) protein is detected in this PHF [Journal of Biochemistry (Toky
o), 99, 1807-1810 (1986)]. When this phosphorylated tau protein is degraded with a protease, various phosphopeptides are obtained, and it is expected that examination of these phosphopeptides will help elucidate Alzheimer's disease in the future.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to clarify the relationship between tau protein phosphorylation and Alzheimer's disease.

[0004]

In order to achieve the above object, it is necessary to synthesize a peptide in which a specific site is phosphorylated, and to prepare an antibody using the peptide. Therefore, the present inventors have attempted to produce some phosphopeptides containing specific phosphorylation sites in tau protein, and have reached the present invention. That is, the gist of the present invention is the formula in claim 1.
It resides in the novel phosphopeptide represented by (1).

Hereinafter, the present invention will be described in detail. The symbols used in the following description indicate the following, respectively. MBHA resin: p-methylbenzhydrylamine resin; Boc: t-butyloxycarbonyl group; Bzl: benzyl group; R: phenyl group which may be substituted by an alkyl group having 1 to 4 carbon atoms; Tos: p-
Toluenesulfonyl group; 2-ClZ: 2-chlorobenzyloxycarbonyl group.

The phosphopeptide of the present invention is composed of 12 amino acids arranged in the order shown in the formula (1) in claim 1, and PO (OH) is added to the OH group of the 10th L-serine (Ser) in the sequence. ) It has a structure in which _two groups are bonded. To produce the phosphopeptide of the present invention, a well-known solid phase peptide synthesis method is applied. For example, an MBHA resin is set in an automatic peptide synthesizer, and Boc-Arg (Tos) -OH, Boc-Pro-O, which have been prepared in advance,
H, Boc-Ser [PO (OR) ₂ ] -OH, Boc
-Thr (Bzl) -OH, Boc-Asp-OH, B
oc-Gly-OH, Boc-Ser (Bzl) -O
H, Boc-Val-OH, Boc-Val-OH, B
oc-Pro-OH, Boc-Ser (Bzl) -O
H and Boc-Lys (2-ClZ) -OH are supplied to sequentially condense. Then, the resulting side chain is protected H-Lys (2-ClZ) -Ser (Bzl) -P
ro-Val-Val-Ser (Bzl) -Gly-A
sp-Thr (Bzl) -Ser [PO (OR) ₂ ]-
The Pro-Arg (Tos) -MBHA resin is reacted with hydrogen fluoride to separate the peptide from the MBHA resin, and then hydrolyzed to convert the PO (OR) ₂ group into PO (OH) _2.
You can change it to the base.

Boc-Ser [PO (OR) ₂ ] -OH used in the present invention
The serine derivative represented by, for example, a phenol represented by HO-R was reacted with phosphoryl chloride (POCl ₃ ) to obtain Cl-PO (OR) ₂ , which was then reacted with Boc-Ser-OBzl. Thereafter, the benzyl group was removed by hydrogenolysis to remove Boc-Ser [PO
(OR) ₂ ] —OH can be easily produced.

[0008]

The novel phosphopeptide of the present invention is expected to be used as a diagnostic agent for Alzheimer's-type senile dementia by detecting PHF using an antibody against the same.

[0009]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which, however, are not intended to limit the scope of the invention.

Example 1 Preparation of Cl-PO (OR) ₂ : (a) 17.5 ml (188 mmol) of phosphoryl chloride, 0.16 g (6.6 mmol) of magnesium chips and 2-methylphenol 31
ml (300 mmol) were mixed and heated under reflux in a 120 ° C. oil bath. After heating at 120 ° C for 1 hour, the temperature of the oil bath was raised to 180 ° C and heating was continued for 3 hours. The resulting viscous oil was then distilled to give a bis [(2-
Methylphenyl) oxy] phosphoryl chloride was obtained in 67% yield.

(B) 2- (a) used in (a) of Example 1
Except that 2-t-butylphenol was used in place of methylphenol, the same treatment as in (a) was carried out to give a boiling point of 185 to 190 ° C / 0.2.
mmHg of bis [(2-t-butylphenyl) oxy] phosphoryl chloride was obtained in 43% yield. (c) Except that 2,6-dimethylphenol was used in place of 2-methylphenol used in (a) of Example 1 above, the same treatment as in (a) was carried out and the boiling point was 173 ° C./0.2 mmHg screw
[(2,6-Dimethylphenyl) oxy] phosphoryl chloride was obtained in 53% yield.

Example 2 Preparation of Boc-Ser [PO (OR) ₂ ] -OBzl: (a) In a pyridine solution containing 5 g (16.9 mmol) of Boc-Ser-OBzl, 1.1 equivalents of the obtained bis [(2-methylphenyl) oxy] phosphoryl chloride were added at 0 ° C.
Stirred for 2-20 hours. After diluting the reaction mixture with 200 ml of ethyl acetate, 2M hydrochloric acid (200 ml × 2) and brine (200 ml ×
Washing was performed sequentially in 3). The organic layer was separated, dried, and dried over anhydrous sodium sulfate.The solvent was removed.The residual oil was purified by column chromatography using ethyl acetate as a solvent, recrystallized from hexane, and Boc-Ser [PO ( OR) ₂ ] -OBzl (R: 2-
Methylphosphonic acid derivative 69)
% Yield. Mass spectrum (MS) and NM of this substance
R was as follows. MS 454 (100%), 555 (M +, 17.9%); 1 H
-NMR (CDCl ₃ ), 1.42 (s, 9H), 2.20 (s, 6H), 4.54 (ddd, J = 9.5,
6.0,2.9 Hz, 1H), 4.58 (dtd, J = 8.0,2.9,2.8 Hz, 1H), 4.65
(ddd, J = 9.5,6.0,2.9 Hz, 1H), 5.05 (d, J = 12.2 Hz, 1H), 5.1
5 (d, J = 12.2 Hz, 1H), 5.35 (d, J = 8.0 Hz, 1H), 7.06-7.27 (m,
8H), 7.31 (m, 5H)

(B) using bis [(2-t-butylphenyl) oxy] phosphoryl chloride in place of bis [(2-methylphenyl) oxy] phosphoryl chloride used in (a) of Example 2 above; and Treated in the same manner as in (a) except that a catalytic amount of dimethylaminopyridine (DMAP) was added, and shown as oily Boc-Ser [PO (OR) ₂ ] -OBzl (R: 2-t-butylphenyl). The resulting serine derivative was obtained in 68% yield. MS of this substance
And NMR were as follows. MS 482 (100%), 583 (M ⁺ , 0.3
%); ¹ H-NMR (CDCl ₃ ), 1.37 (s, 9H), 1.38 (s, 9H), 1.40 (s, 9H),
4.53 (m, 2H), 4.63 (ddd, J = 9.6,5.1,2.6 Hz, 1H), 4.92 (d, J =
12.2 Hz, 1H), 5.11 (d, J = 12.2 Hz, 1H), 5.28 (d, J = 7.9 Hz, 1
H), 7.11 (dddd, J = 7.9,6.2,5.0,1.8 Hz, 2H), 7.16 (dddd, J =
7.9,6.2,6.2,1.8 Hz, 2H), 7.23 (m, 2H), 7.3 (m, 3H), 7.36 (d
ddd, J = 7.9,4.6,1.8,1.8 Hz, 2H), 7.50 (dd, J = 16.7,7.9 H
z, 2H)

(C) using bis [(2,6-dimethylphenyl) oxy] phosphoryl chloride instead of bis [(2-methylphenyl) oxy] phosphoryl chloride used in (a) of Example 2; Except for the addition of a catalytic amount of DMAP and the same treatment as in (a), the oily Boc-Ser [PO (OR) ₂ ] -OBzl
(R: 2,6-dimethylphenyl) represented by the serine derivative 76
% Yield. The MS and NMR of this material were as follows. MS 482 (100%), 583 (M +, 0.3%); 1 H-NMR (CDCl 3), 1.42
(s, 9H), 2.28 (s, 6H), 2.29 (s, 6H), 4.46 (m, 2H), 455 (ddd, J =
9.3,5.8,2.5 Hz, 1H), 5.02 (d, J = 12.3 Hz, 1H), 5.05 (d, J =
8.6Hz, 1H), 5.12 (d, J = 12.3Hz, 1H), 7.06 ((m, 6H), 7.31 (m,
5H)

Example 3 Production of Boc-Ser [PO (OR) ₂ ] -OH: (a) A serine derivative obtained in (a) of Example 2 in ethyl acetate and a palladium catalyst (Pd / C) and stirred in a hydrogen stream at room temperature for 2 hours. The reaction mixture was filtered using celite, and the filtrate was concentrated under reduced pressure to give an oil.
A serine derivative represented by Boc-Ser [PO (OR) ₂ ] -OH (R: 2-methylphenyl) was obtained in a quantitative yield. The MS and NMR of this product were as follows. MS 276 (100%), 465 (M +, 2.8%); 1 H-NMR
(CDCl ₃ ), 1.42 (s, 9H), 2.19 (s, 3H), 2.21 (s, 3H), 4.55 (m, 2
H), 4.67 (m, 1H), 5.54 (d, J = 7.6 Hz, 1H), 6.30 (br.s, 1H), 7.
07 (m, 2H), 7.13 ((t, J = 7.3 Hz, 2H), 7.18 (t, J = 7.3 Hz, 2H),
7.25 (d, J = 5.6 Hz, 2H)

(B) A serine derivative represented by Boc-Ser [PO (OR) ₂ ] -OBzl (R: 2-t-butylphenyl) instead of the serine derivative used in (a) of Example 3 above Was treated in the same manner as in Example 3 (a) except that Boc-Ser [PO (OR) ₂ ] -OH
A serine derivative represented by (R: 2-t-butylphenyl) was obtained in a quantitative yield. The MS and NMR of this product were as follows. M
S475 (100%), 549 ( M +, 2.5%); 1 H-NMR (CDCl 3), 1.31 (s, 9H),
1.36 (s, 9H), 1.38 (s, 9H), 4.28 (br.s, 1H), 4.36 (m, 1H), 4.6
7 (m, 1H), 5.20 (br.s, 1H), 5.54 (d, J = 6.7 Hz, 1H), 7.05 (dt,
J = 7.6,7.5 Hz, 2H), 7.12 (dt, J = 14.0,7.5 Hz), 7.32 (dd, J =
16.0,7.6 Hz, 2H), 7.46 (dd, J = 25.8,7.5 Hz, 2H)

(C) Instead of the serine derivative used in (a) of Example 3, Boc-Ser [PO (OR) ₂ ] -OBzl (R: 2,6-
The same treatment as in (a) of Example 3 was carried out except that the serine derivative represented by dimethylphenyl) was used, and Boc-Ser [PO (O (O
A serine derivative represented by R) ₂ ] -OH (R: 2,6-dimethylphenyl) was obtained in a quantitative yield. The MS and NMR of this product were as follows. MS 392 (100%), 493 (M +, 6%); 1 H-NMR (CDCl 3), 1.
42 (s, 9H), 2.26 (s, 3H), 2.27 (s, 3H), 4.37 (m, 1H), 4.45 (m, 1
H), 4.53 (m, 1H), 5.23 (d, J = 7.5 Hz, 1H), 6.98 (m, 6H), 7.89
(br.s)

Example 4 (a) H-Lys (2-ClZ) -Ser (Bzl) -Pro-Val-Val-Ser (Bzl) -Gl
Production of y-Asp-Thr (Bzl) -Ser [PO (OR) ₂ ] -Pro-Arg (Tos) -MBHA resin: 0.94 g of MBHA resin (amine content 0.64 mmol / g resin)
Was set on a Biosearch 9500-type automatic peptide synthesizer, and Boc-Arg (Tos) -OH, Boc-Pro-OH, Boc-Ser [PO
(OR) ₂ ] -OH (R: 2-methylphenyl), Boc-Thr (Bzl) -OH, Bo
c-Asp-OH, Boc-Gly-OH, Boc-Ser (Bzl) -OH, Boc-Val-O
H, Boc-Val-OH, Boc-Pro-OH, Boc-Ser (Bzl) -OH and Boc-
Lys (2-ClZ) -OH was supplied to sequentially condense to obtain the above side chain-protected peptide-MBHA resin.

(B) Hydrogen fluoride treatment: 1 g of the side chain-protected peptide-MBHA resin obtained in the above (a) was collected and set in a hydrogen fluoride reaction device manufactured by Peptide Research Institute, Protein Research Promotion Association. Then, it was reacted with 10 ml of hydrogen fluoride at 0 ° C. for 1 hour in the presence of 1.5 ml of anisole. After completion of the reaction, hydrogen fluoride was distilled off under reduced pressure.
The residue was washed with ethyl acetate and then 100 ml of 2M acetic acid.
Extraction processing, and H-Lys-Ser-Pro-Val
-Val-Ser-Gly-Asp-Thr-Ser
285 mg of a crude peptide having a protected phosphate group represented by [PO (OR) ₂ ] -Pro-Arg-NH ₂ was obtained.

This was dissolved in 20 ml of 30% acetic acid and applied to a column of Sephadex G-25 (inner diameter 5 cm, length 100 cm), and eluted with the same solvent to collect a fraction containing the target compound. . Next, this was dissolved in a small amount of distilled water, and purified by HPLC (high-performance liquid chromatography) using a reverse phase column (inner diameter 2 cm, length 25 cm) in which ODS (octadecylsilane) was bonded to silica. . Elution is 5 in 30% in 0.1% trifluoroacetic acid
Performed by applying a concentration gradient of acetonitrile from% to 65%. The yield of the purified product was 144 mg. The mass spectrum (FAB-MS) of this substance and the amino acid ratio in the acid hydrolyzate were as follows. FAB-MS [M + H] + 1489, calcd _{(C 65 H 102 N 17 O} 21 P + H) 1489; Asp 0.99 (1), Thr 0.93
(1), Ser 2.66 (3), Pro 1.71 (2), Gly 1.10 (1), Val
1.89 (2), Lys 1.10 (1), Arg 1.00 (1)

(C) Hydrogenolysis: 60 mg of the peptide protected with a phosphate group obtained in (b) above and 100 mg of platinum oxide (catalyst) are combined with 1
After mixing with ml of acetic acid and stirring for 12 hours at room temperature under a hydrogen pressure of 5-6 atm, the catalyst was filtered off. The filtrate is lyophilized, purified by preparative HPLC, and the final product, H-Lys-Ser-Pro-
34 mg of a phosphopeptide represented by Val-Val-Ser-Gly-Asp-Thr-Ser [PO (OH) ₂ ] -Pro-Arg-NH ₂ was obtained. The structure of this substance was confirmed by the following FAB-MS, amino acid analysis and sequence analysis. FAB-MS [M + H] ⁺ 1309, calculated value (C ₅₁ H ₉₀ N ₁₇ O
₂₁ P + H) 1309; Asp 0.96 (1), Thr 1.08 (1), Ser 2.60
(3), Pro 1.98 (2), Gly 1.21 (1), Val 2.11 (2), Lys
1.24 (1), Arg 1.00 (1)

[Sequence list] SEQ ID NO: 1 Sequence length: 12 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence characteristics: Ser at No. 10 in the sequence binds to _two PO (OH) groups ing. Array:

Claims (1)

(57) [Claims] [Claim 1] The following formula (1) [Wherein X represents a PO (OH) ₂ group].