JP2916296B2 - 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.

[0006] The novel phosphopeptide of the present invention comprises 13 amino acids arranged in the order shown in formula (1) in claim 1, and L-serine (Ser) at position 6 in the sequence.
And a structure in which a PO (OH) ₂ group is bonded to at least one OH group of the ninth L-threonine (Thr). To produce these phosphopeptides, a well-known solid phase peptide synthesis method is applied. For example, in order to produce a phosphopeptide in which X in the formula (1) is a PO (OH) ₂ group and Y is a hydrogen atom, an MBHA resin is set in an automatic peptide synthesizer, and B
oc-Arg (Tos) -OH, Boc-Ser (Bz
l) -OH, Boc-Gly-OH, Boc-Pro-
OH, Boc-Thr (Bzl) -OH, Boc-Gl
y-OH, Boc-Pro-OH, Boc-Ser [P
O (OR) ₂ ] -OH, Boc-Gly-OH, Boc
-Pro-OH, Boc-Ser (Bzl) -OH, B
oc-Ser (Bzl) -OH and Boc-Lys (2
—ClZ) —OH is supplied to sequentially condense. Then
The resulting side chain is protected H-Lys (2-Cl
Z) -Ser (Bzl) -Ser (Bzl) -Pro-
Gly-Ser [PO (OR) ₂ ] -Pro-Gly-
Thr (Bzl) -Pro-Gly-Ser (Bzl)
An Arg (Tos) -MBHA resin may be reacted with hydrogen fluoride to separate the peptide from the MBHA resin, and then hydrolyzed to convert PO (OR) ₂ groups to PO (OH) ₂ groups.

In order to produce a phosphopeptide of the formula (1) wherein both X and Y are PO (OH) ₂ groups, Boc-Thr (Bzl) -OH used in the above item 0006 is replaced by Boc-Thr (Bzl) -OH. -Thr [PO (O
R) ₂ ] -OH may be used in the same manner as in paragraph 0006. Further, in the formula (1), X is a hydrogen atom, and Y is PO (OH)
To produce a phosphopeptide of _two groups, the above 00
Boc-Ser [PO (OR) ₂ ] -OH
The treatment may be performed in the same manner as in paragraph 0006 except that -Ser (Bzl) -OH is used and Boc-Thr [PO (OR) ₂ ] -OH is used instead of Boc-Thr (Bzl) -OH.

The Boc-Ser [PO (OR) ₂ ] used in the present invention
Amino acid derivatives represented by -OH and Boc-Thr [PO (OR) 2] -OH , for example, phenol represented by HO-R - by reacting Le acids into phosphoryl chloride _{(POCl 3) Cl-PO (} OR ) _2, and after reacting it with Boc-Ser-OBzl or Boc-Thr-OBzl, removing the benzyl group by hydrogenolysis to give Boc-Ser
It can be easily produced by using [PO (OR) ₂ ] -OH or Boc-Thr [PO (OR) ₂ ] -OH.

[0009]

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

[0010]

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 viscous oil thus obtained was distilled to give bis [(2-methylphenyl) oxy] phosphoryl chloride having a boiling point of 145 to 150 ° C./0.03 mmHg in a 67% yield.

(B) 2 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 and the boiling point was 185-190 ° 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 and Boc-Thr [PO (OR) ₂ ] -OBzl: (a) 5 g (16.9 mmol) of Boc-Ser-OBzl 1.1 equivalents of bis [(2-methylphenyl) oxy] phosphoryl chloride obtained in (a) of Example 1 was added to the pyridine solution containing the pyridine solution at 0 ° C.
Stirred for ~ 20 hours. After the reaction mixture was diluted with 200 ml of ethyl acetate, it was successively washed with 2M hydrochloric acid (200 ml × 2) and brine (200 ml × 3). After separating and collecting the organic layer and drying over anhydrous sodium sulfate, the solvent was removed, and the residual oil was purified by column chromatography using silica gel using ethyl acetate-hexane as a solvent, recrystallized with hexane and Boc-Ser [PO (O
A serine derivative represented by the formula (R) ₂ ] -OBzl (R: 2-methylphenyl) was obtained in a 69% yield. Mass spectrum (MS) of this substance
And NMR were as follows. MS454 (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, 1
H), 5.15 (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) Bis [(2-t-butylphenyl) oxy] phosphoryl chloride is used in place of bis [(2-methylphenyl) oxy] phosphoryl chloride used in (a) of Example 2 above, The same treatment as in (a), except that a catalytic amount of dimethylaminopyridine (DMAP) was added, followed by oily Boc-Ser [PO (OR) ₂ ] -OBzl (R: 2-t-butylphenyl) The indicated serine derivative was obtained in 68% yield. Of this substance
MS 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, 9
H), 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, 1H), 7.11 ((dddd, J = 7.9,6.2,5.0,1.8 Hz, 2H), 7.16 (d
ddd, J = 7.9,6.2,6.2,1.8 Hz, 2H), 7.23 (m, 2H), 7.3 (m, 3H),
7.36 (dddd, J = 7.9,4.6,1.8,1.8 Hz, 2H), 7.50 (dd, J = 16.7,
(7.9 Hz, 2H)

(C) using bis [(2,6-dimethylphenyl) oxy] phosphoryl chloride in place of bis [(2-methylphenyl) oxy] phosphoryl chloride used in (a) of Example 2 above, Except for adding a catalytic amount of DMAP and treating in the same manner as in (a), the oily Boc-Ser [PO (OR) ₂ ] -OB
A serine derivative represented by zl (R: 2,6-dimethylphenyl)
Obtained in 76% yield. The MS and NMR of this material were as follows. MS 482 (100%), 583 (M + 0.3%); 1 H-NMR (CDCl 3), 1.4
2 (s, 9H), 2.28 (s, 6H), 2.29 (s, 6H), 4.46 (m, 2H), 4.55 (ddd,
J = 9.3,5.8,2.5 Hz, 1H), 5.02 (d, J = 12.3 Hz, 1H), 5.05 (d, J
= 8.6 Hz, 1H), 5.12 (d, J = 12.3 Hz, 1H), 7.06 (m, 6H), 7.31
(m, 5H)

Example 3 Preparation 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 at room temperature for 2 hours in a hydrogen stream. The reaction mixture was filtered using celite, and the filtrate was concentrated under reduced pressure to give an oily Boc-
A serine derivative represented by Ser [PO (OR) ₂ ] -OH (R: 2-methylphenyl) was obtained in a quantitative yield. Its MS and NMR 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) Instead of the serine derivative used in (a) of Example 3 above, Boc-Ser [PO (OR) ₂ ] -OBzl (R: 2-t-
Butylphenyl), and treated in the same manner as in Example 3 (a), except that the serine derivative represented by Boc-Ser [PO (O (O
A serine derivative represented by the formula: R) ₂ ] -OH (R: 2-t-butylphenyl) was obtained in a quantitative yield. The MS and NMR of this product were as follows. MS 475 (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), 4.67 (m, 1H), 5.20 (br.s, 1H), 5.54 (d, J = 6.7 Hz, 1
H), 7.05 (dt, J = 7.6,7.5 Hz, 2H), 7.12 (dt, J = 14.0,7.5 H
z), 7.32 (dd, J = 16.0,7.6 Hz, 2H), 7.46 (dd, J = 25.8,7.5 H
z, 2H)

(C) Instead of the serine derivative used in (a) of Example 3 above, 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) -Ser (Bzl) -Pro-Gly-Ser [PO
(OR) ₂ ] -Pro-Gly-Thr (Bzl) -Pro-Gly-Ser (Bzl) -Arg (Tos)-
Production of MBHA resin: 0.94 g of MBHA resin (amine content 0.64 mmol
/ G resin) in a 9500 type automatic peptide synthesizer manufactured by Biosearch, Inc., and Boc-Arg (Tos) -OH, Boc-Ser (Bzl) -O
H, Boc-Gly-OH, Boc-Pro-OH, Boc-Thr (Bzl) -OH, Boc-Gl
y-OH, Boc-Pro-OH, Boc-Ser [PO (O (O
R) ₂ ] -OH (R: 2-methylphenyl), Boc-Gly-OH, Boc-Pro-O
H, Boc-Ser (Bzl) -OH, Boc-Ser (Bzl) -OH and Boc-Lys (2-C
lZ) -OH was supplied to sequentially condense to obtain 2.5 g of the above side chain-protected peptide-MBHA resin.

(B) Hydrogen fluoride treatment: 0.5 g of the side chain-protected peptide-MBHA resin obtained in (a) above was collected, and this was used as a hydrogen fluoride reaction apparatus manufactured by Peptide Research Institute, Protein Research Promotion Association. And reacted with 10 ml of hydrogen fluoride at 0 ° C. for 1 hour in the presence of 1 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-Ser-Pro
-Gly-Ser [PO (OR) ₂ ] -Pro-Gly
It was obtained -Thr-Pro-Gly-Ser- Arg-NH 2 with crude peptide 135mg protecting the phosphate groups represented.

This was dissolved in 20 ml of 30% acetic acid, 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 65 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] + 1474, calcd _{(C 63 H 97 N 18 O} 21 P + H) 1474; Thr0.93 (1), Ser 3.1 (4),
Pro 2.7 (3), Gly 3.0 (3), Lys 0.98 (1), Arg 1.0
(1)

(C) Hydrogenolysis: 60 mg of the peptide having a protected 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 and purified by preparative HPLC to give the final product, H-Lys-Ser-Ser-
Pro-Gly-Ser [PO (OH) ₂ ] -Pro-Gly-Thr-Pro-Gly-Ser-Arg-N
To obtain a phosphopeptide 35 mg represented by H _2. The structure of this substance was confirmed by the following FAB-MS, amino acid analysis and sequence analysis. FAB-MS [M + H] + 1294, calcd (C ₄₉ H ₈₅ N ₁₈
O ₂₁ P + H) 1294; Thr 1.10 (1), Ser 3.3 (4), Pro 3.4
(3), Gly 2.9 (3), Lys 1.02 (1), Arg 1.06 (1)

Example 5 Boc-Ser (Bzl) -OH was used in place of Boc-Ser [PO (OR) ₂ ] -OH (R: 2-methylphenyl) used in (a) of Example 4. And, instead of Boc-Thr (Bzl) -OH, a known Boc-Thr [PO (OR) ₂ ]-
Except for using OH (R: phenyl), after treating in the same manner as in (a) of Example 4, subsequently treating in the same manner as in (b) and (c) in Example 4, H-Lys- Ser-Ser-Pro-Gly-Ser-Pro-Gly-Thr
A phosphopeptide represented by [PO (OH) ₂ ] -Pro-Gly-Ser-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] ⁺
1294, calcd _{(C 49 H 85 N 18 O} 21 P + H) 1294; Thr 0.97 (1), Se
r3.95 (4), Pro 2.90 (3), Gly 2.94 (3), Lys 1.08
(1), Arg 1.00 (1)

Example 6 Boc-Thr (Bzl) -OH used in Example 4 (a) was replaced with B
After the same treatment as in (a) of Example 4 except that oc-Thr [PO (OR) ₂ ] -OH (R: phenyl) was used,
H-Lys-Ser-Ser-Pro-Gly-
Ser [PO (OH) ₂ ] -Pro-Gly-Thr [PO (OH) ₂ ] -Pro-Gly-Ser-Arg-
A phosphopeptide represented by 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] + 1373, calcd (C ₄₉ H ₈₆ N ₁₈ O
₂₄ P ₂ + H) 1374; Thr 0.73 (1), Ser 3.12 (4), Pro 2.60
(3), Gly 3.00 (3), Lys 1.16 (1), Arg 1.00 (1)

[Sequence list]

SEQ ID NO: 1 Sequence length: 13 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence characteristics: At least one of Ser at position 6 and Thr at position 9 in the sequence is PO (OH ) It is bonded to _two groups.

Claims (1)

(57) [Claims] [Claim 1] The following formula (1) [Wherein, X and Y each represent a PO (OH) ₂ group or a hydrogen atom,
And at least one of X and Y represents a PO (OH) ₂ group].