JPH04346998A - Chain peptide and its production - Google Patents

Abstract

PURPOSE:To obtain a new chain peptide capable of converting a metal into a ligand for elucidating the function of a cysteine-containing protein according to a fragment condensing method, etc., and providing a hydrogenase model having biological analogous function. CONSTITUTION:A compound expressed by the formula HCl.H-Pro-OMe (Me is methyl) is reacted with a compound expressed by Boc-Cys(Acm)-OH (Boc is tert-butyloxycarbonyl; Acm is acetamidomethyl) in DMF according to a fragment condensing method to provide a compound expressed by the formula Boc-Cys(Acm)-Pro-OMe. The C-terminal ester group is then hydrolyzed and subsequently reacted with a compound expressed by the formula HCl.H-Leu-Cys(Acm)-OMe in the presence of a condensing agent in DMF to afford the compound expressed by formula I. The resultant compound is further reacted with mercury chloride in dimethyl sulfoxide to provide a mercury-containing peptide expressed by formula II, which is then reacted with hydrogen sulfide to eliminate the mercury as mercury sulfide. Thereby, the objective chain peptide expressed by formula III is obtained.

Description

[Detailed description of the invention]

0001

TECHNICAL FIELD The present invention relates to a novel chain peptide, a metal complex thereof, a method for producing the same, and intermediates used in the production.

0002

BACKGROUND OF THE INVENTION In recent years, the specificity of metal complexes has attracted attention in a wide range of fields such as medicines, diagnostic agents, and functional materials. For example, in the pharmaceutical field, cisplatin, a platinum complex, is known as an anti-cancer drug, and a type of gold mercaptan complex is known as an effective anti-rheumatic drug. Several other active complexes have also been reported. In addition, efforts are being made to develop catalysts that have biologically similar functions by utilizing their oxidation-reduction ability.

[0003] Furthermore, peptides themselves are attracting attention for their physiological activity. For example, gramicidin S, a cyclic peptide, has strong antibacterial activity, and recently many analogs of this, their synthesis methods, and activities have been reported.

On the other hand, as research on metalloproteins progresses, the state of the active center (metal binding site) is gradually clarified along with technical and theoretical advances in inorganic chemistry, and the amino acid sequence of the active center is used to express the function of metalloproteins. It is gradually becoming clear that they play an important role in However, cysteine-containing proteins have not been studied in detail.

[0005]

[Problems to be Solved by the Invention] The present invention provides a means for elucidating the function of cysteine-containing proteins, and creates a novel cysteine-containing peptide that can be used as a hydrogenase model with biologically similar functions by liganding it with a metal. Its task is to provide this information.

[0006]

[Means for Solving the Problems] The inventors of the present invention have conducted intensive research to produce a cysteine-containing peptide metal complex that satisfies the above requirements, and have discovered the following facts.

1) A typical globular water-soluble metalloprotein has hydrophilic amino acids arranged on the outside and hydrophobic amino acids arranged on the inside. This hydrophobic environment maintains various electrostatic interactions within the protein and gives the protein its enzymatic function. Therefore, when synthesizing a model complex that can cause electrostatic interactions, it is necessary to ensure that the metal binding site is hydrophobic and is not affected by external solvents.

2) Cysteine-containing metalloproteins have two types of chelate ligands, and the metal binding site of alcohol dehydrogenase has two independent Cys-X-Y-Cys type sequences with D2d symmetry and Zn(II ), Cy
The s-X-Y-Cys-A-B-Cys type sequence is chelated to Zn(II) with C2 symmetry,
Both ZnS4 cores have an almost tetrahedral structure. Therefore, a similar sequence is appropriate.

3) For example, in the simulation of the active site of nickel thiolate complexes as part of research on nickel hydrogenase, the redox potential of various allylthiolate and alkylthiolate (RS-) nickel complexes was investigated. , the nickel complex that allows Ni(III) to exist stably is [Ni(SR)4]
It was expected to be 2-.

[0010] In view of the above experimental facts, and for the following reasons, the present inventor has developed a hydrogenase model as follows:
New formula (1) ◆ Boc-Cys-Pro-Leu-Cys-OMe
...(1)◆ (In the formula, Boc is an amino group protected with a t-butyloxycarbonyl group, Cys is cysteine, Pro is proline, Leu is leucine, and OMe is a carboxy group protected with a methoxy group). The indicated linear peptide was obtained.

[0011] The novel peptide of formula (1) of the present invention (hereinafter referred to as C
PLC) has a Cys-X-Y-Cys type sequence and contains hydrophobic amino acids such as proline and leucine, so it can reproduce the electrostatic interaction of the metal binding site of a metalloprotein.

[0012] Furthermore, in the CPLC of the present invention, the amino group and carbonyl group at the terminal of the peptide, which may be involved in metal coordination, are protected with a Boc- group and -OMe group, which are hydrophobic protecting groups. be.

[0013]P. Balaram et al.'s study (J. Am. Chem. Soc., 105, 7423 (
1983). ) from CPLC has type III in the molecule
It is expected that the CPL of the present invention will have a β-turn structure.
It can be expected that C easily coordinates with metals.

[0014] Research by Nakamura et al. [Bull. Chem. So
c. Jpn. 56, 3647 (1983) and Bi
ochim. Biophys. Acta, 788, 30
6 (1984) and Inorg. Chem. 24
, 2190 (1985). and Inorg. Chem
．． 26, 1978 (1987). ] shows a quasi-reversible redox potential close to that of rubredoxin [Fe(Z-C
Although ys-Pro-Leu-Cys-OMe)2]2- was selected as a model complex, it is thought that the CPLC of the present invention has a similar effect.

Furthermore, since the CPLC of the present invention does not contain an aromatic nucleus in its molecule, amide protons clearly appear during 1H-NMR analysis, making it easy to analyze the conformation of the CPLC main chain skeleton.

The present invention provides: ◆1) a novel linear peptide (CPLC) represented by the above formula (1); 2) a method for producing the same; ◆3) the following novel intermediate used in the production method: b) Boc-Cys-(Acm)-Pro
-OMe...(2)◆(In the formula, Acm represents the SH group of cysteine protected with an acetamidomethyl group. The same applies hereinafter.)◆b) Boc-Cys-(Acm
)-Pro-OH...(3)◆c)
Boc-Cys-(Acm)-Pro-
Leu-Cys(Acm)-OMe
…(4)◆d) Boc-Cys-
(HgCl)-Pro-Leu-Cy
s(HgCl)-OMe...(
5) ◆ and ◆4) A novel chain peptide (CPLC) represented by formula (1) and gold, silver, cadmium, nickel, palladium, ruthenium, mercury, zinc, molybdenum,
CPL obtained by reacting with a metal selected from the group consisting of cobalt, iron, titanium, manganese, silicon and calcium or a compound thereof.
The present invention provides a metal complex of C and a method for producing the same.

[0017]

Effects of the Invention The chain peptides and metal complexes thereof of the present invention are biosimilar compounds and have metal trapping ability and metal transport ability in living bodies, so they can be effectively used as medicines, diagnostic agents, and functional materials. be done. The inventor is
After considering the CPLC synthesis route, we decided to adopt the fragment condensation method. The advantages of this method are 1) a small number of reaction steps, 2) ease of product purification because the properties of the used fragment and the generated peptide are different, and 3) the ability to freely select the size of the fragment. 4) If several types of fragments are synthesized, they can be used for other peptide synthesis. The disadvantage of this method is that there is a risk of racemization during condensation, but L-proline is selected as the C-terminus of the acid component, and DCC (dicyclohexylcarbodiimide)-HOBt (1-hydroxybenzotriazole) is used as the condensing agent. This can be solved by using Furthermore, it is preferable to select, as the protecting group, a Boc group, an OMe group, and an Acm group, which are protecting groups that are not removed under mutual deprotection conditions.

[0018]

[Examples] The present invention will be explained in detail with reference to Examples below.

Example 1◆ 1) Peptide ligand C by fragment condensation method
Synthesis of PLC (1) (see Figure 1) ◆ A) Boc-Cys(Acm)-Pro-OMe(
2) Synthesis ◆ HCl・H-Pro-OMe (1.66 g, 10 mm
ol) and 100 ml of dimethylformamide (hereinafter referred to as D
(abbreviated as MF) and cooled to -3°C with N-
Methylmorpholine (1.10ml, 10mmol) was added. After 10 minutes, Boc-C was added to the colorless and translucent reaction solution.
ys(Acm)-OH (2.92g, 10mmol)
, 1-hydroxybenzotriazole (hereinafter abbreviated as HOBt) (1.62 g, 12 mmol), dicyclohexylcarbodiimide (hereinafter abbreviated as DCC) (2
．． 27 g, 11 mmol) were added in this order, and the mixture was stirred at -3°C for 5 hours, at 0°C for 12 hours, and at room temperature for 5 hours. After distilling off DMF in vacuo, ethyl acetate was added and cooled, the precipitated crystals were separated by filtration, the filtrate was washed successively with an aqueous sodium bicarbonate solution, an aqueous citric acid solution, and water, and then dried over sodium sulfate to remove the solvent. The resulting oil was purified by silica gel column chromatography to obtain 3.45 g (yield: 85.5%) of the desired product (2) as a colorless and translucent oil. Its characteristics were as follows.

[α]D = −98.9° (c 0.68, MeOH
, 21.6°C) ◆ 1H-NMR (CDCl3) 1
00MHz◆δTMS (ppm)=1.44
(s, 9H, Boc CH3)◆ 1.76-2.18 (m, 4H, Proβ, γ
-H)◆2.02 (s, 3H, A
cm CH3)◆2.80~3.00 (m
, 2H, Cys β-H)◆3.62-3.82
(m, 2H, Proδ-H)◆3.72
(s, 3H, OCH3)◆4.24~4.
66 (m, 4H, Acm CH2, ◆Pr
o α-H, Cys α-H) ◆ 5.50 (d, H, Cys N
H)◆7.02~7.24 (m,H,Acm
NH)◆b) Boc-Cys(Acm)-Pro
Synthesis of -OH (3) ◆ (2) (3.45 g, 8.5 g in 85 ml methanol)
5 mmol) and 1M sodium hydroxide aqueous solution (
12.83 ml, 12.83 mmol) was added thereto, and the mixture was stirred at room temperature for 3.5 hours. After methanol was distilled off in vacuo, 85 ml of water was added, 50 ml of 10% citric acid aqueous solution was added to adjust the pH to 3, and the target product was extracted with ethyl acetate. The organic phase was washed with water, dried, and the solvent was distilled off in vacuo to give a colorless translucent oil (3), 2.02 g (yield: 60
．． 7%). Its characteristics were as follows.

[α]D = −53.1° (c 0.67, MeOH
, 23.0°C). ◆ 1H-NMR (DMSO-d6) 100 Hz◆δ
DSS (ppm)=1.40 (
s, 9H, Boc CH3)◆ 1.76-2.08 (m, 4H, Pro β
, γ-H)◆1.86 (s, 3H
, Acm CH3)◆2.58~2.88
(m, 2H, Cys β-H)◆3.52-3.72
(m, Pro δ-H)◆4.08~4.4
4 (m, 4H, Acm CH2, ◆Pro
α-H, Cys α-H)◆ 7.06 (d,H,Cys N
H)◆8.36~8.56 (t,H,Acm
NH)◆c) Boc-Cys(Acm)-Pro
-Synthesis of Leu-Cys(Acm)-OMe(4) ◆H
Cl・H-Leu-Cys(Acm)-OMe(1.8
N-methylmorpholine (0.5 g, 5.2 mmol) was dissolved in 70 ml of DMF and cooled to -3°C.
572 mmol, 5.2 mmol) were added. 10
Minutes later (2) (2.02g, 5.2mmol), HO
Bt (0.838g, 6.2mmol), DCC (1
．． 18 g, 5.7 mmol) were added one after another, and the mixture was stirred at -3°C for 5 hours, at 0°C for 15 hours, and at room temperature for 30 hours. D
MF was distilled off in vacuo, and the remaining solution was dissolved in ethyl acetate, cooled, and the precipitate was filtered off. Add the filtrate to aqueous sodium hydrogen carbonate solution,
It was washed with a citric acid aqueous solution and water and dried. The solvent was distilled off in vacuo, and the resulting crude product was purified by silica gel column chromatography to obtain 1.15 g (yield 42.0%) of the desired product (4) as a white powder. Its characteristics were as follows.

[α]D = −91.71° (c 0.67, Me
OH, 23.0°C) ◆ 1H-NMR (CDCl3) 100 Hz ◆ δTM
S (ppm) = 0.80 to 1.04 (m, 6
H, Leu δ-H)◆ 1.42 (s, 9H, Boc
CH3)◆1.56~1.68 (m, 3H,
Leu β-H, Leu γ-H) 1.84 to 2.12 (m, 4H, Pro β
-H, Pro γ-H) 2.02 to 2.04 (s, 6H, Acm C
H3)◆2.84~3.08 (m, 4H, C
ys β-H)◆3.68~3.88 (m,
2H, Pro δ-H)◆3.74
(s,3H,OCH3)◆4.18~4.28
(m, H, Leu α-H)◆4.30~4.
44 (m, 4H, Acm CH2)◆4.
44-4.84 (m, 3H, Pro α-H
, Cys α-H) 5.44 (d,H,Cys(1)
NH)◆7.00~7.28 (m, 2H
,Leu NH,Cys(2) NH) 7.40~7.64 (m,2H,Acm N
H)◆d) Boc-Cys(HgCl)-Pro-
Synthesis of Leu-Cys(HgCl)-OMe (5) ◆Mercury chloride (1.18 g, 4.35 mmol) was dissolved in 4 ml of dimethyl sulfoxide (hereinafter abbreviated as DMSO). This solution was added to the DMSO solution of (4) with stirring, and stirred at room temperature for 5 hours. Water was added, and the white crystals formed were filtered off using a glass filter and washed with water. The crystals were transferred to a Schlenk and vacuum dried to give 1.34 g (yield: 90
．． 7%) of the target product (5) was obtained. Its characteristics were as follows.

[α]D = +2.91° (c 0.68, MeOH
,28.2℃)◆ 1H-NMR (DMSO-d6)
500 Hz◆δ(ppm)=0.81
(d, 3H, Leu δ-H)◆ 0.88 (d, 3H, Leu
δ-H)◆1.37 (s, 9H,
Boc CH3 )◆1.52~1.58 (
m, 2H, Leu β-H)◆1.60-1.64
(m, H, Leu γ-H)◆1.79-1.
85 (m, H, Pro β-H)◆1.85
~1.93 (m, 2H, Pro γ-H)◆
2.21-2.26 (m, H, Pro β-
H)◆3.10~3.15 (m,H,Cys(
2) β-H)◆3.22 (
d, H, Cys (1) β-H)◆3.25
(d,H,Cys(2) β-H)
◆3.47~3.5 (m, H, Cys(1)
β-H)◆3.60~3.64 (m,H
, Pro δ-H)◆3.62
(s,3H,OCH3)◆3.77~3.81
(m, H, Pro δ-H)◆4.24-4.28
(m, H, Leu α-H)◆4.28~4
．． 32 (m, H, Pro α-H)◆4.5
0 to 4.54 (m, H, Cys(2) α
-H)◆4.70~4.73 (m,H,Cys
(1) α-H)◆6.88
(m, H, Cys(1) NH)◆7.67
(d, H, Leu NH)◆7.75
(d, H, Cys(2) N
H) ◆13C-NMR (DMSO-d6) 500 M
Hz◆δ(ppm)=20.66 (
C, Leu δ-C)◆23.00
(C, Leu δ-C)◆24.50
(C, Leu γ-C) ◆24.61
(C, Pro γ-C) ◆28.05
(3C, Boc CH3)◆28.
26 (C,Cys(1) β-
C)◆29.31 (C, Cys(2
) β-C)◆29.43 (C
, Pro β-C) ◆39.73
(C, Leu β-C) ◆47.26
(C, Pro δ-C) ◆51.34
(C, Leu α-C) ◆52.07
(C, OCH3) ◆54.26
(C, Cys(1) α-C)◆55.
84 (C, Cys(2) α-
C)◆61.62 (C,Pro
α-C)◆78.65 (C, Boc
C-O)◆154.73 (C,
Boc C=O)◆170.17 ~170.48(
3C, Cys (1) C=O, Pro C=O,
◆Leu C=O)◆ 173.15 (C, Cys(2)
C=O)◆E) Boc-Cys-Pro-Le
Synthesis of u-Cys-OMe (1) (CPLC) ◆ All the following operations were performed under an argon atmosphere.

(5) (410g, 0.402mmol
) and dissolve it by adding 70 ml of methanol from which air has been removed.
Hydrogen sulfide was bubbled in while stirring at room temperature. At this time,
Black crystals of mercury sulfide formed. Gravity filtration is performed using a glass filter, the crystals are washed with methanol from which air is removed, and the solvent is vacuum distilled from the colorless and transparent filtrate and dried.
mg (yield 63.7%) of the target product (1) as a white powder was obtained. Its characteristics were as follows.

1H-NMR (DMSO-d6) 500 MHz◆
δ (ppm) = 0.82 ~ 0.91 (m, 6H
,Leu δ-H)◆ 1.37 (s,9H,Boc
CH3)◆1.45~1.50 (m, 2H,
Leu β-H)◆1.61~1.71 (m
, H, Leu γ-H)◆1.76-1.83
(m, H, Pro β-H)◆1.83-1.93
(m, 2H, Pro γ-H)◆2.00~
2.10 (m, H, Pro β-H)◆2.
53-2.63 (m, 2H, Cys(2)
β-H)◆2.72~2.89 (m, 2H,
Cys(1) β-H)◆3.59-3.71
(m, H, Pro δ-H)◆3.63
(s, 3H, OCH3)◆4.26~4
．． 38 (m, 3H, Leu α-H, Pro
α-H, ◆Cys (2) α-H) ◆4.41
~4.47 (m, H, Cys(1) α-H)
◆7.11 (d, H, Cys(1
) NH)◆8.00 (d,
H, Leu NH)◆8.19
(d,H,Cys(2)NH)◆Example 2◆ Reaction of CPLC with NiCl2.6H2O (complex formation by triethylamine method) All operations were performed under an argon stream.

1) Preparation of titration curve ◆ Measurement of the ultraviolet-visible spectrum was carried out using a Hitachi 228A photometer using a quartz cell with a thickness of 1 mm.

a) Solution preparation ◆ b) CPLC (22.0
mg, 40 μmol) was dissolved in methanol, toluethylamine (11.2 μl, 80 μmol) was added, and 4
．． A 0mM CPLC solution was prepared.

B) NiC in a 10ml volumetric flask
A 4.0 mM solution was prepared by dissolving 12.6H2O (9.5 mg, 40 μmol) in methanol.

b) Titration test◆a) 4.0mM Ni in 4.0mM CPLC solution
A fixed amount of Cl2.6H2O solution was added and mixed by shaking for about 1 minute, and then the brown solution was transferred to a quartz cell and the ultraviolet-visible spectrum was measured.

B) 324nm and 420nm
The extinction coefficient of the peak was plotted. As a result, a clear inflection was observed at a ratio of [CPLC]/[Ni] of about 2 at all wavelengths. This is CPLC2
This shows that one Ni atom is liganded in the molecule.

2) Measurement of 1H-NMR ◆ In the same manner as the titration, CPLC and NiCl2.6H2O are reacted in the presence of triethylamine to form a complex. After distilling off the solvent and drying in vacuum, about 10 mg of the sample is It was dissolved in heavy methanol from which air had been removed, and 1H-NMR was measured. The results were as follows.

1H-NMR (CD3 OD) 500 MHz◆δ(
ppm) = 0.90 to 0.99 (m, 6H, L
eu δ-H)◆ 1.26-1.30 (m, 9H, Boc C
H3)◆1.30~1.34 (t,9H,N
Et3 CH3)◆1.57~1.68 (
m, 2H, Leu β-H)◆1.68-1.80
(m, H, Leu γ-H)◆1.94-2.
04 (m, 2H, Pro β-H)◆2.1
0-2.16 (m, 2H, Pro γ-H)
◆2.81~2.98 (m, 2H, Cys(2
) β-H)◆3.15~3.45 (m,
2H, Cys(1) β-H)◆3.17-3.2
4 (q, 6H, NEt3 CH2)◆3.
63-3.80 (m, 2H, Pro δ-H
)◆3.73 (m, 3H, OCH
3)◆4.28~4.37 (m, H, Leu
, α-H)◆4.37~4.49 (m,H
, Pro, α-H)◆4.54~4.60
(m, H, Cys(2) α-H)◆4.60~4
．． 65 (m, H, Cys(1) α-H)
◆Example 3◆ Reaction of CPLC with NiCl2.6H2O (complex formation by sodium hydride method)◆ 1) Creation of titration curve◆ a) Solution preparation◆ a) CPLC (21
．． 4mg, 39.0μmol) was dissolved in methanol, and 7.8mM sodium hydride/methanol solution was added to 1.5mg, 39.0μmol).
46 ml was added to prepare a 3.9 mM CPLC solution.

B) NiC in a 10 ml volumetric flask
l2 ・6H2 O (9.3 mg, 39 μmmol)
was dissolved in methanol to prepare a 3.9mM solution.

b) Titration test◆a) 3.9mM Ni in 3.9mM CPLC solution
A fixed amount of the Cl2.6H2O solution was added and shaken for about 1 minute, and then the brown solution was transferred to a quartz cell and the ultraviolet-visible spectrum was measured.

(b) The extinction coefficients of the peaks at 324 nm and 420 nm were plotted. As a result, an inflection was observed at a ratio of [CPLC]/[Ni] of about 2 at all wavelengths. This is CPLC2 molecule and Ni
This shows that one molecule is a ligand for each atom.

[Brief explanation of drawings]

[Figure 1] Schematic diagram showing an example of the method for producing chain peptides of the present invention

Claims (9)

[Claims] [Claim 1] The following formula (1) Boc-Cys-Pro-Leu-Cys-OMe
...(1) (In the formula, Boc is an amino group protected with a t-butyloxycarbonyl group, Cys is cysteine, Pro is proline, Leu is leucine, and OMe is a carboxy group protected with a methoxy group). chain peptide. 2. The method for producing a chain peptide according to claim 1, which comprises using a fragment condensation method. [Claim 3] The following formulas (2), (3), (4), (5)
Boc-Cys(Acm)-Pro-OMe...(2
) Boc-Cys(Acm)-Pro-OH...(3
) Boc-Cys(Acm)-Pro-Leu-Cys
(Acm)-OMe...(4) Boc-Cys(HgCl)-Pro-Leu-Cys
(HgCl)-OMe...(5) (In the formula, Boc is an amino group protected with a t-butyloxycarbonyl group, Cys is cysteine, Acm is an SH group of cysteine protected with an acetamidomethyl group, Pr
Claim 2 characterized in that chain peptides represented by o is proline, Leu is leucine, and OMe is a carboxy group protected with a methoxy group are sequentially used as intermediates.
Manufacturing method described. [Claim 4] The following formula (2) Boc-Cys(Acm)-Pro-OMe...(2
) (In the formula, Boc is an amino group protected with a t-butyloxycarbonyl group, Cys is cysteine, Acm is an SH group protected with an acetamidomethyl group, Pro is proline, and OMe is a carboxy group protected with a methoxy group. A chain peptide represented by [Claim 5] The following formula (3) Boc-Cys(Acm)-Pro-OH...(3)
(In the formula, Boc is an amino group protected with a t-butyloxycarbonyl group, Cys is cysteine, Acm is an SH group protected with an acetamidomethyl group, and Pro is a proline). [Claim 6] The following formula (4) Boc-Cys(Acm)-Pro-Leu-Cys(
Acm)-OMe...(4) (In the formula, Boc is an amino group protected with a t-butyloxycarbonyl group, Cys is cysteine, Acm is an SH group of cysteine protected with an acetamidomethyl group, Pr
o is proline, Leu is leucine, and OMe is a carboxy group protected with a methoxy group). [Claim 7] The following formula (5) Boc-Cys(HgCl)-Pro-Leu-Cys
(HgCl)-OMe...(5) (In the formula, Boc is an amino group protected with a t-butyloxycarbonyl group, Cys is cysteine, Pro is proline, Leu is leucine, and OMe is a carboxy group protected with a methoxy group. A chain peptide represented by 8. The chain peptide according to claim 1, gold,
silver, cadmium, nickel, palladium, ruthenium,
A method for producing a chain peptide metal complex, which comprises reacting a metal selected from the group consisting of mercury, zinc, molybdenum, cobalt, iron, titanium, manganese, silicon, and calcium or a compound of the metal. 9. A chain peptide metal complex produced by the production method according to claim 8.