JP2003183298A - A liquid-phase peptide synthesis method in which amino acids are sequentially added by a compatible-multiphase organic solvent system - Google Patents

Abstract

(57) [Summary] (Modified) [Problem] To provide a liquid phase peptide synthesis technique that can be substituted for a solid phase reaction peptide synthesis method. [Solution] A state of compatibility and a state of phase separation by controlling temperature. The peptide is synthesized using a solvent system that can reversibly control the state. The amino acid residue to be introduced is converted into the C of the peptide to be synthesized using a carrier that increases the solubility in one of the solvents A constituting the solvent system
A peptide starting compound in which a terminal amino acid residue is bonded to a carrier and amino acids are sequentially introduced into the compound. The other solvent B
At a temperature below the temperature at which the compatible state is formed, various amino acids used for elongation of the peptide chain are preferentially dissolved, and at a temperature at or above the compatible state, the solvent in a state compatible with the solvent A Is used to dissolve the peptide starting compound, and the solvent B in which various protected amino acids are dissolved is replaced with a solution in which amino acids that synthesize sequentially designed peptides in a state of phase separation are dissolved, By heating, the amino acids are sequentially bonded.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to oligomers or polymers such as peptides, proteins, DNAs, RNAs and polysaccharides, especially peptides, and one or more kinds of unit molecules constituting them, especially amino acids. It relates to a method of synthesizing said oligomers or polymers by combining them in order, in particular a liquid phase peptide synthesis method. The present invention relates to a liquid phase peptide synthesis method using a solvent system in which the synthesis of specifically designed oligomers or polymers, particularly peptides, can be easily controlled and the reaction products can be easily recovered.

[0002]

2. Description of the Related Art In a chemical reaction, if a desired product can be easily separated from a reagent, a catalyst, a reaction aid, a by-product, etc., not only can the separation operation be significantly reduced, but also the process The amount of chemicals used can be reduced, and the generation of wastes harmful to the environment can be suppressed as much as possible. By the way, so far, peptides and DN
Oligomers and polymers such as A are synthesized by sequentially binding and extending a plurality of unit compounds according to the design. For the synthesis, a functional group capable of forming oligomers and polymers to be synthesized on a solid surface, for example, in the case of a peptide, an amino acid residue at the carboxy terminal of the peptide introduced on the surface of an insoluble resin was dispersed in a solvent. A solid-phase synthesis method for synthesizing peptides in the state has been used. However, in this reaction system, since the chemical reaction must be performed on the solid surface, the reagent is difficult to approach the solid surface, and the reactivity and the reaction rate are generally low. Further, there is a problem that it is not possible to easily confirm that the desired reaction has proceeded. Further, in solid phase synthesis, it is difficult to increase the reaction scale, and many solid phase carriers are expensive, which is problematic in terms of economy.

[0003]

SUMMARY OF THE INVENTION Therefore, the object of the present invention is to provide a solution peptide synthesis method of the above-mentioned prior art, which realizes the separation of the obtained reaction product very easily while the solid phase surface reaction is a liquid phase reaction. Is to establish. In the course of intensive studies to solve the above problems, a solvent system developed by the present inventors, which can reversibly control a compatible state and a phase-separated state by controlling the temperature, was developed. It was considered to establish a method for synthesizing peptides using the same. For that purpose, it has a binding part of an amino acid unit that initiates peptide formation in the conventional solid-phase reaction peptide synthesis method, has a function of sequentially binding amino acid units and carrying an extended peptide chain, and further starts the synthesis of the peptide. It is necessary to find a compound residue having a function of dissolving a compound having a binding part of the preceding amino acid unit and a compound having a peptide chain being bound during synthesis in one solvent or a mixed solvent system. In the present specification, it is called a carrier because of the technical characteristics required for the compound residue. Then, as one of the solvents or the mixed solvent A, one composed of a nonpolar organic solvent is used.
Liquid phase peptide synthesis method system learned from the solid phase reaction peptide synthesis method by using the compound of the general formula A as a carrier to be used in combination with another solvent or a solvent system consisting of a polar organic solvent as the mixed solvent B. It was possible to solve the above problems.

[0004]

The present invention relates to a method for synthesizing a peptide using a solvent system capable of reversibly controlling the compatible state and the phase separated state by controlling the temperature. As a residue for introducing an amino acid residue at the carboxy terminus of the peptide to be used, a carrier derived from a compound that enhances solubility in one of the solvents or the mixed solvent A that constitutes the solvent system capable of controlling the state is used, A combination of a solvent or mixed solvent A and the carrier was used to extend the peptide chain by sequentially introducing amino acids into the peptide starting compound in which the carboxy-terminal amino acid residue of the peptide to be synthesized is bound to the carrier and the peptide starting compound. Another solvent which enhances the solubility of the compound in the solvent or the mixed solvent A, and which is combined with the solvent or the mixed solvent A, or As the mixed solvent B, various amino acids used for elongation of the peptide chain are preferentially dissolved at a temperature not higher than the temperature at which the compatible state is formed, and compatible with A at a temperature higher than the temperature at which the compatible state is formed. A peptide that is sequentially designed in a phase-separated state using the above-mentioned B in which a protected amino acid having a protecting group bonded to various α-position amino groups is dissolved using a solvent that forms a solvent in the state to dissolve the peptide starting compound. Is a liquid phase peptide synthesis method characterized in that the amino acid to be synthesized is replaced with a dissolved amino acid, and the amino acid is sequentially bonded by heating to a temperature at which a compatible state is exhibited after the replacement. Preferably, one solvent or mixed solvent A comprises an organic solvent, and the other solvent or mixed solvent B combined with the solvent or mixed solvent A also comprises an organic solvent, the liquid phase peptide synthesis method, More preferably, one of the solvents or the organic solvent constituting the mixed solvent A is a cycloalkane compound, and the other solvent or the organic solvent constituting the mixed solvent B is combined with the organic solvent constituting the mixed solvent A. Is a liquid phase peptide synthesis method, characterized in that it is composed of at least one selected from the group consisting of nitroalkane, nitrile, alcohol, alkyl halide, amide compound and sulfsulfoxide, More preferably, the alkyl group of the nitroalkane has 1, 2 or 3 carbon atoms and is a nitrile Has an alkyl group having 1, 2 or 3 carbon atoms, and the amide compound has an alkyl group of N-dialkyl or N-monoalkylamide and an acyl group or formyl group having a total of 6 or less carbon atoms, and the alcohol has carbon atoms. In the liquid phase peptide synthesis method, the number of which is 8 or less, the alkyl group of sulfoxide has 1, 2 or 3 carbon atoms, and the alkyl group of the halogenated alkyl has 6 or less carbon atoms. Yes, and even more preferably, the carrier forming the peptide starting compound has a functional group that binds to the parent cycloalkane-based solvent moiety and the amino acid, and the aromatic carbon atom represented by the general formula A is characterized. A method for synthesizing each of the above-mentioned liquid-phase peptides, which comprises a residue from a basic skeleton compound of a hydrogen ring or a hydrocarbon group having 10 or more carbon atoms, Properly is the liquid phase peptide synthesis method, wherein the one represented by the compound group B.

In the conventional solid phase reaction peptide synthesis method, since the amino acid binding group is present in the solid phase, the reactivity with the amino acid is poor, and the reaction on the solid surface is completed by supplying a large excess amount of the amino acid to the reaction system. There was a problem that I had to do. However, in the liquid phase peptide synthesis method of the present invention, since the peptide synthesis reaction proceeds in a homogeneous solution system in a compatible state, the reaction efficiency is extremely high, and the amino acid binding group bound to the carrier is externally added. No excess amount of amino acid molecule is required to react.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail. A. The solvent system of the present invention comprises at least two or more single organic solvents capable of reversibly taking a state of a homogeneously compatible mixed solvent system and a state of a separated solvent system separated into a plurality of phases by a slight temperature change, or The mixed solvent consists of a mixed organic solvent, and one of the organic solvents or the mixed organic solvent dissolves the peptide starting compound and the compound in which an amino acid is sequentially bound to the peptide starting compound in the state of a separated solvent system and the extended peptide chain is bound. The other organic solvent or mixed organic solvent that does not dissolve the amino acid to be dissolved dissolves the amino acid to be bound in the state of the separated solvent system, but binds the peptide starting compound and the amino acid sequentially bound thereto to the extended peptide chain. Basically, it has the property of not dissolving the compound.

The one organic solvent or the mixed organic solvent is selected on the basis of the above-mentioned basic characteristics, but is basically a low-polarity organic solvent, and the group of compounds constituting the solvent is alkane. , Cycloalkanes, alkenes, alkynes, aromatic compounds, and the like. Preferable ones are cycloalkane-based compounds, and cyclohexane is particularly preferable. It is speculated that the solvent system of the present invention has been realized in connection with the conversion of the chair-boat conformer of cyclohexane under relatively mild temperature conditions in relation to other solvents. can do. Cyclohexane has a relatively high melting point of 6.5 ° C., and has the advantage of being capable of solidifying and separating the product after the reaction, and also from this aspect, it can be said that it is a preferable solvent.

The single organic solvent or the mixed organic solvent to be combined with 2, the above 1 is selected on the basis of the above basic characteristics as in the case of 1, but is basically a highly polar organic solvent. Preferably, nitroalkane, nitrile,
It is selected from the group consisting of alcohols, alkyl halides, amide compounds and sulfoxides.

3. The solution phase peptide synthesis method of the present invention is established in combination with the above-mentioned solvent system in order to improve the solubility of the peptide starting compound in one single organic solvent or a mixed organic solvent in the state of a separated solvent system. It is important to select one that is insoluble in the other single organic solvent or the mixed organic solvent combined with the one single organic solvent or the mixed organic solvent, and as such, in the following general formula A It is selected from the residues shown and residues from a basic skeleton compound of a hydrocarbon group having 10 or more carbon atoms.

[0010]

[Chemical 4]

In the general formula A, L ₁ is a single bond which is bonded to a hydroxyl group, a thiol group, an amino group or a carbonyl group which is bonded to an amino acid, and an atomic group which is bonded to the hydroxyl group, a thiol group, an amino group or a carbonyl group. , Or an atomic group that forms a fused aromatic ring having two rings by combining with a dotted line, and a dotted line represents an atomic group that forms a condensed aromatic ring by combining with H or with L _1. Is O, S, N,
An ester group, a sulfide group or an imino group, R
Is O, which enhances the solubility in cycloalkane-based solvents,
It is a hydrocarbon group having 10 or more carbon atoms, which may contain S or N as a bonding atom. n is an integer of 1 to 5.

Specific examples of the general formula A include compounds of the following general formula group B.

[0013]

[Chemical 5]

In each general formula, X, R and n are the same as in general formula A. Q is a single bond or a hydrocarbon group, and R is
₂ is a hydroxyl group, a thiol group, an amino group or a carbonyl group which binds to an amino acid, and R ₃ and R ₄ are groups of the following general formula C.

[0015]

[Chemical 6]

R ₅ is a hydroxyl group, a thiol group, an amino group or a carbonyl group which binds to an amino acid.

4. The amino acid used in the liquid phase peptide synthesis method of the present invention is a protected amino acid used in the conventional solid phase reaction peptide synthesis method, for example, Fmoc (9-fluorenylmethoxycarbonyl) -amino acid, Boc ( T-butoxycarbonyl) -amino acid, Cbz (benzyloxycarbonyl) -amino acid and the like can be used.

Here, more specific examples will be shown as examples, but this is for the purpose of making the present invention easier to understand and does not limit the present invention.

Example 1 Soluble carrier [SC] -valine (Val) -glycine (G
ly) -phenylalanine (Phe) -Fmoc [(S
C) -Val-Gly-Phe-Fmoc)]. As a soluble carrier [SC], in the general formula group B, R is C ₁₈ H ₃₇ - is, X is O, n is 3, Q is CH _2, R ₂ is OH, ( 3,4,5-trioctadecyloxyphenyl) methan-1-ol,
[(3,4,5-trioctadecyloxyphenyl) methan-1-ol] is used.

Step 1) Fmoc-Val (170 mg)
Is dissolved in 3 mL of dichloromethane, and 125 mg of dicyclohexylcarbodiimide (DCC) is added.
The solution was stirred at room temperature for 15 minutes and then filtered. The filtrate is concentrated to dryness with a rotary evaporator, and the obtained residue is dissolved in 3 mL of dimethylformamide (DMF). Subsequently, 3 mL of a cyclohexane solution (soluble carrier 50 mg / 3 mL) in which the soluble carrier [SC] was dissolved was added to DMF.
Add to solution. 4-dimethylaminopyridine (D
MAP) 6.5 mg is added, the reaction solution is heated to 50 ° C., and the reaction is performed for 30 minutes. At this time, cyclohexane layer and D
The solution system separated into the MF layer becomes a uniform solution system. After completion of the reaction, the reaction solution is returned to room temperature, and the reaction solution is separated into two phases again. The lower DMF phase is separated and removed, 3 mL of 10% diethylamine / DMF solution is added, and the mixture is stirred at 50 ° C. for 20 minutes. The reaction solution is cooled and the cyclohexane layer is separated. Soluble carrier-bound valine-NH ₂ ([SC] -Val-NH ₂ ) is recovered in this cyclohexane layer.
(Yield 95%)

Step 2) Fmoc-Gly 57 mg, HO
Bt55mg, diisopropylcarbodiimide (DIPC
D) Dissolve 25 mg in 2 mL DMF and stir for 150 minutes at room temperature. This solution was activated with Fmoc-glycine-OH
/ Used as a DMF solution. That is, [SC] -Val-NH obtained in step 1) after cooling 2 mL of this solution to 5 ° C.
₂ / Cyclohexane solution (2 mL) is added. The reaction solution is gently raised from 5 ° C to 50 ° C over 1 hour,
Further, it is left at 50 ° C. for 30 minutes. Finally, when the reaction solution is cooled to room temperature, it is separated into two layers again, so that the desired product [SC] -Val-G is obtained from the upper layer (cyclohexane layer).
ly-Fmoc) is separated. The Fmoc group was eliminated by adding diethylamine to the same solution [SC] -Va.
1-Gly-NH ₂ is obtained. FIG. 1 shows the outline of this synthetic reaction.

Step 3) Next, Fmoc-Phe57m
g, HOBt 55 mg, and diisopropylcarbodiimide (DIPCD) 25 mg are dissolved in DMF 2 mL, and the mixture is stirred for 150 minutes at room temperature. This solution is used as an activated Fmoc-phenylalani (Phe) -OH / DMF solution. That is, 2 ml of this solution was cooled to 5 ° C., and then the [SC] -Val-Gly-NH ₂ / cyclohexane solution (2 mL) obtained in step 2) was added. The reaction solution is gently raised from 5 ° C. to 50 ° C. over 1 hour, and left at 50 ° C. for 30 minutes. Finally, cool the reaction to room temperature and reapply.
Since it is separated into two layers, the desired product [SC] -Val-Gly-Phe-Fmo is obtained from the upper layer (cyclohexane layer).
c) is separated. By repeating the above operation, amino acids are sequentially bound to the soluble carrier, and the target oligopeptide is synthesized.

Structural confirmation [SC] cyclohexane soluble carrier; (3,4,5-trioctadecyloxyphenyl) methan-1-ol [(3,4,5-trioctadecyloxyphenyl) methan-1-ol]. ¹ H-NMR (400 MHz) δ; 5.54 (2H,
s), 4.58 (2H, d, J = 5.1Hz), 3.9.
6 (4H, t, J = 6.6Hz), 3.96 (3H,
s), 1.82-1.70 (6H, m), 1.50-
1.41 (6H, m), 1.38-1.20 (84H,
br), 0.88 (9H, 6,8Hz), ¹³ C-NMR
(100 Hz); (100 MHz δ: 153.2, 1
37.4, 136.0, 105.2, 73.4, 69.
1, 65.7, 32.0, 30.4, 29.8, 29.
7, 29.5, 26.2, 22.8, 14.2; MA
LDI TOF-MS (pos), calculated for C ₆₁ H ₁₁₆ O ₄ [M + Na] ⁺ 935, experimental 935.

[SC] -Val-Fmoc; ¹ H-NMR (CDCl ₃ ) δ 7.76 (2H, d, J
= 7.7 Hz), 7.60 (2H, d, J = 7.7H)
z), 7.40 (2H, dt, J = 2.6, 7.3H)
z), 7.31 (2H, t, J = 7.3 Hz), 6.5
3 (2H, s), 5.31 (1H, d, J = 9.2H
z), 5.11 (1H, d, J = 12.1 Hz), 5.
05 (1H, d, J = 12.1 Hz), 4.38 (2
H, m), 4.23 (1H, t, J = 7.3 Hz),
3.94 (6H, m), 2.19 (1H, m), 1.7
8 (4H, m), 1.73 (2H, m), 1.45 (6
H, m), 1.35-1.23 (84H, br.),
0.95 (3H, d, J = 7.0Hz), 0.88 (1
2H, m), ¹³ C-NMR (CDCl ₃ ) δ172.
0, 156.2, 153.2, 143.9, 143.
8, 141.3, 138.4, 130.2, 128,
3, 127.7, 127.1, 125.1, 120.
0, 107.1, 73.4, 69.2, 67.4, 6
7.1, 59.0, 47.2, 32.0, 31.4, 3
0.3, 29.8, 29.7, 29.5, 29.4, 2
6.1, 22.7, 14.1; TOF-MS (pos)
Calculated value for MF, C ₈₁ H ₁₃₅ NO ₇ [M + Na] ⁺ 1
257, experimental value 1257

[SC] -Val-NH₂ ¹ H-NMR (400 MHz) δ: 6.54 (2H,
s), 5.07 (1H, d, J = 12.1Hz), 50
3 (1H, d, J = 12.1 Hz), 3.95 (4
H, t, J = 6.6 Hz), 3.94 (2H, t, J
= 6.6 Hz), 3.33 (2H, d, J = 5.1H)
z), 2.07-2.01 (1H, m), 1.81-
1.77 (4H, m), 1.76-1.71 (2H,
m), 1.49-1.43 (6H, m), 1.37-
1.23 (84H, br), 0.96 (3H, d, J =
7.0 Hz), 0.89-0.86 (12H,
m) ,;¹³C-NMR (150 MHz) δ, 175.
4, 153.2, 138.3, 130.7, 107.
1, 73.4, 69.2, 66.8, 59.9, 32.
2, 32.0, 30.3, 29.8, 29.7, 29.
6, 29.4, 26.1, 22.7, 19.3, 17.
1, 14.1; TOF-MS (pos) C₆₆H₁₂₅NO_Five
[M + Na] ⁺Calculated value 1034, experimental value 103
Four

[SC] -Val-Gly-Fmoc ¹ H-NMR (400 MHz) δ: 7.77 (2H,
d, J = 7.3 Hz), 7.59 (2H, d, J =
7.3 Hz), 7.40 (2H, t, J = 7.3H)
z), 7.31 (2H, dt, J = 0.7, 7.3H)
z), 6.52 (2H, s), 6.38 (1H, d, J
= 8.4 Hz), 5.44-5.37 (1H, b
r), 5.10 (1H, d, J = 12.1 Hz),
5.02 (1H, d, J = 12.1 Hz), 4.62
(2H, dd, J = 8.4, 4.8 Hz), 4.42
(2H, d, J = 7.0 Hz), 4.24 (1H,
t, J = 7.0 Hz), 3.96-3.92 (8H,
m), 2.21-2.16 (1H, m), 1.81-
1.76 (4H, m), 1.75-1.70 (2H,
m), 1.48-1.43 (6H, m), 1.37-
1.21 (84H, br), 0.91 (3H, d, J =
7.0 Hz), 0.88 (9H, t, J = 7.0H
z), 0.86 (3H, d, J = 7.0 Hz), ¹³
C-NMR (150 MHz) δ: 171.5, 16
8.7, 156.5, 153.1, 143.6, 14
1.2, 138.3, 130.0, 127.7, 12
7.0, 125.0, 120.0, 107.0, 73.
4, 69.2, 67.5, 67.4, 57.1, 47.
1, 32.0, 31.4, 30.4, 29.8, 29.
7, 29.5, 29.4, 26.1, 22.8, 19.
0, 17.7, 14.2; MALDI TOF-MS
(Pos) C ₈₃ H _{1 38} N ₂ O ₈ [M + Na] ⁺ calculated value 1314, experimental value 1314

[SC] -Val-Gly-NH ₂ ¹ H-NMR (600 MHz) δ: 7.74 (1H,
d, J = 9.2 Hz), 6.53 (2H, s), 5.
11 (1H, d, J = 12.1 Hz), 5.02 (1
H, d, J = 12.1 Hz), 4.61 (1H, d
d, J = 9.2, 5.1 Hz), 3.95 (4H,
t, J = 6.6 Hz), 3.94 (2H, t, J =
6.6 Hz), 3.39 (2H, s), 2.24-
2.18 (1H, m), 1.81-1.76 (4H,
m), 1.75-1.71 (2H, m), 1.49-
1.44 (6H, m), 1.37-1.20 (84H,
br), 0.93 (3H, d, J = 7.0 Hz),
0.90-0.86 (12H, m); ¹³ C-NMR
(150 MHz) δ: 172.6, 171.8, 15
3.1, 130.3, 125.5, 106.9, 73.
4, 69.2, 67.2, 56.6, 44.8, 32.
0, 31.3, 30.4, 30.3, 29.8, 29.
7, 29.5, 29.4, 26.2, 22.8, 19.
1, 17.8, 14.2; MALDITOF-MS (p
os) Calculated value for C ₆₈ H ₁₂₈ N ₂ O ₆ [M + Na] ⁺ 1
091, experimental value 1091

[SC] -Val-Gly-Phe-Fmoc ¹ H-NMR (600 MHz) δ: 7.75 (2H,
d, J = 7.7 Hz), 7.53-7.49 (2H,
m), 7.39 (2H, dd, J = 7.3, 2.2H
z), 7.30-7.27 (4H, m), 7.25-
7.21 (1H, m), 7.20-7.15 (2H, b
r), 6.76-6.69 (1H, br), 6.60-
6.55 (1H, br), 6.50 (2H, s), 5.
40-5.34 (1H, br), 5.07 (1H, d,
J = 12.1 Hz), 4.99 (1H, d, J = 1)
2.1 Hz), 4.56 (1H, dd, J = 8.8,
4.8 Hz), 4.46-4.30 (2H, m),
4.17 (1H, t, J = 7.0 Hz), 4.10-
4.03 (1H, m), 3.92 (6H, t, J = 6.
6 Hz), 3.83-3.76 (2H, m), 3.1
8-3.11 (1H, m), 3.10-3.02 (1
H, m), 2.20-2.13 (1H, m), 1.79.
-1.69 (6H, m), 1.48-1.41 (6H,
m), 1.35-1.23 (84H, br.m), 0.
91-0.85 (15H, m); ¹³ C-NMR (150
MHz) δ: 171.5, 171.3, 168.3,
156.0, 153, 1, 143.6, 141.2, 1
38.2, 136.2, 130.1, 129.1, 12
8.8, 127.7, 127.1, 127.0, 12
5.0, 124.9, 120.0, 107.0, 73.
4, 69.2, 67.5, 67.1, 57.3, 47.
2, 32.0, 31.3, 30.4, 29.8, 29.
7, 29.5, 29.4, 26.2, 22.8, 19.
0, 17.8, 14.2; MALDI TOF-MS
(Pos) C ₉₂ H ₁₄₇ N ₃ O ₉ [M + Na] ⁺ calculated value 1461, experimental value 1461

[SC] -Val-Gly-Phe-NH ₂ ¹ H-NMR (400 MHz) δ: 7.99-7.93
(1H, m), 7.35-7.30 (2H, m), 7.
27-7.21 (3H, m), 6.66 (1H, d, J
= 8.8 Hz), 6.52 (2H, s), 5.11
(1H, d, J = 12.1), 5.02 (1H, d, J
= 12.1 Hz), 4.58 (1H, dd, J = 8.
8, 4.8 Hz), 4.05 (1H, d, J = 5.9)
Hz, minor), 4.01 (1H, d, J = 5.9H
z, major), 3.98-3.91 (7H, m), 3.
66 (1H, d, J = 10.0 Hz), 3.32 (1
H, dd, J = 13.6, 3.9 Hz), 2.67
(1H, dd, J = 13.6, 10.0 Hz), 2.
24-2.15 (1H, m), 1.82-1.69 (6
H, m), 1.50-1.39 (6H, m), 1.37
-1.21 (84H, br), 0.92 (3H, d, J
= 6.8 Hz), 0.90-0.85 (12H,
m); ¹³ C-NMR (150 MHz) δ: 175.
2, 171.5, 168.9, 153.1, 151.
4, 137.7, 129.2, 128.8, 126.
9, 125.5, 107.0, 73.5, 69.2, 6
7.5, 57.2, 56.5, 43.4, 40.9, 3
2.0, 31.3, 30.4, 29.8, 29.7, 2
9.5, 29.4, 26.2, 22.8, 19.1, 1
7.7, 14.2; MALDI TOF-MS (po
s) Calculated value for C ₇₇ H ₁₃₇ N ₃ O ₇ [M + Na] ⁺ 12
39, experimental value 1239

Example 2 [SC] -valine-phenylalanine-Fmoc ([SC] -Va
Liquid-phase synthesis of l-Phe-Fmoc) Fmoc-Phe (63 mg), HOBt (63 mg) and diisopropylcarbodiimide (DIPCD) (25 mg) were dissolved in DMF (2 mL), and the mixture was stirred at room temperature for 150 minutes. This solution is used as an activated Fmoc-Phe / DMF solution. That is, 2 mL of this solution was cooled to 5 ° C., and then the [SC] -Val-NH ₂ / cyclohexane solution (2 m) obtained in Step 1) of Example 1 was used.
L) is added. The reaction solution is gently raised from 5 ° C. to 50 ° C. over 1 hour, and left at 50 ° C. for 30 minutes. Finally, when the reaction solution is cooled to room temperature, it is separated into two layers again, and thus the desired product [SC] -Val-Phe-Fmoc) is separated from the upper layer (cyclohexane layer). By repeating the above operation, amino acids are sequentially bound to the soluble carrier, and the target peptide is synthesized.

Example 3 [SC] -valine-proline-Fmoc ([SC] -Val-Pro-
Liquid phase synthesis of Fmoc) Fmoc-Pro 53 mg, HOBt 57 mg, diisopropylcarbodiimide (DIPCD) 25 mg were dissolved in DMF 2 mL,
Stir for 150 minutes at room temperature. This solution was activated Fmoc-P
Used as a ro-OH / DMF solution. Ie 2 m of this solution
After cooling 1 to 5 ° C., [SC] -Va obtained in 1) of Example 1
adding l-NH ₂ / cyclohexane solution (2 ml).
The reaction solution is gently raised from 5 ° C. to 50 ° C. over 1 hour, and left at 50 ° C. for 30 minutes. Finally, when the reaction solution is cooled to room temperature, it is separated into two layers again, so that the desired product [SC] -Val-P is obtained from the upper layer (cyclohexane layer).
ro-Fmoc) is separated. By repeating the above operation, amino acids are sequentially bound to the soluble carrier, and the target peptide is synthesized.

Example 4 Soluble carrier-valine-alanine-Fmoc ([SC] -Val
-Ala-Fmoc) Liquid Phase Synthesis Fmoc-Ala 50 mg, HOBt 53 mg, and diisopropylcarbodiimide (DIPCD) 25 mg are dissolved in DMF 2 mL and stirred for 150 minutes at room temperature. This solution is used as an activated Fmoc-Ala-OH / DMF solution. That is, after cooling 2 mL of this solution to 5 ° C., the [SC] -Val-NH ₂ / cyclohexane solution (2 ml) obtained in step 1) of Example 1 is added. The reaction solution is gently raised from 5 ° C. to 50 ° C. over 1 hour, and left at 50 ° C. for 30 minutes. Finally, cool the reaction to room temperature and reapply 2
Since the layers are separated, the desired product [SC] -Val-Ala-Fmoc) is separated from the upper layer (cyclohexane layer). By repeating the above operation, amino acids are sequentially bound to the soluble carrier, and the target peptide is synthesized.

[0033]

As described above, according to the method of the present invention, as a compound into which a carboxy-terminal amino acid residue of a peptide to be synthesized is introduced, the compound itself and the compound bound to the peptide constitute a reaction solvent system. In combination with the solvent-solubilizing carrier, a liquid-phase peptide synthesis method is provided that is easier to control than the solid-phase reaction peptide synthesis and that the reaction product is easily recovered. Excellent effect is brought about.

[Brief description of drawings]

FIG. 1 is a schematic view of one embodiment of the liquid phase peptide synthesis method of the present invention.

Claims (6)

[Claims] 1. A method for synthesizing a peptide using a solvent system capable of reversibly controlling the compatibility state and the phase separation state by controlling the temperature, the method comprising synthesizing a carboxy terminal of a peptide to be synthesized. As a residue for introducing the amino acid residue of, a combination with a carrier derived from a compound that enhances solubility in one solvent or a mixed solvent A that constitutes a solvent system capable of controlling the above state is used,
By combining the solvent or mixed solvent A and the carrier, a peptide starting compound in which a carboxy-terminal amino acid residue of the peptide to be synthesized is bound to the carrier and amino acids are sequentially introduced into the peptide starting compound to extend the peptide chain. The compound of formula (1) has higher solubility in the solvent or mixed solvent A, and is used as the other solvent or mixed solvent B combined with the solvent or mixed solvent A at a temperature below the temperature at which the compatible state is formed to extend the peptide chain. Various amino acids to be used are preferentially dissolved, and at a temperature equal to or higher than the temperature at which the compatible state is formed, a solvent that is in a compatible state with the A to form a solvent that dissolves the peptide starting compound is used,
The above-mentioned B in which a protected amino acid having a protecting group bonded to various α-amino groups was dissolved was replaced with one in which amino acids for synthesizing peptides designed sequentially in a state of phase separation were dissolved, and after the substitution, the compatible compound was obtained. A method for synthesizing a peptide in a liquid phase, which comprises sequentially binding the amino acids by heating to the temperature to be exhibited. 2. The liquid according to claim 1, wherein one of the solvents or mixed solvent A comprises an organic solvent, and the other solvent or mixed solvent B combined with the solvent or mixed solvent A also comprises an organic solvent. Phase peptide synthesis method. 3. An organic solvent which constitutes one solvent or a mixed solvent A, wherein the organic solvent comprises a cycloalkane-based compound, and which is combined with the organic solvent constituting the solvent or mixed solvent A, and the other solvent or a mixed solvent B. The liquid phase peptide synthesis method according to claim 2, wherein the solvent comprises at least one selected from the group consisting of nitroalkane, nitrile, alcohol, alkyl halide, amide compound and sulfoxide. 4. The nitroalkane alkyl group has a carbon number of
1, 2 or 3, the number of carbon atoms of the alkyl group of the nitrile is 1, 2 or 3, and the amide compound is the total number of carbon atoms of the alkyl group of N-dialkyl or N-monoalkylamide and the acyl group or formyl group. Is 6 or less, the alcohol has 8 or less carbon atoms, the sulfoxide alkyl group has 1, 2, or 3 carbon atoms, and the alkyl halide group has 6 or less carbon atoms. The liquid phase peptide synthesis method according to claim 3. 5. The carrier forming the peptide starting compound is
The following general formula A is characterized in that it has a functional group that binds to the parent cycloalkane solvent part and an amino acid.
The method for synthesizing a liquid phase peptide according to any one of claims 1 to 4, which comprises a residue from a basic skeleton compound of an aromatic hydrocarbon ring or a hydrocarbon group having 10 or more carbon atoms represented by. [Chemical 1] [In the general formula A, L ₁ is a hydroxyl group bonded to an amino acid, a thiol group, an amino group, or a single bond bonded to a carbonyl group, an atomic group bonded to the hydroxyl group, a thiol group, an amino group, or a carbonyl group, or It is an atomic group that forms a two-ring condensed aromatic ring by combining with the dotted line, and the dotted line is H
X is O, S, N, an ester group, or an atomic group forming a condensed aromatic ring by bonding with or with L ₁ .
It is a sulfide group or an imino group, and R is a hydrocarbon group having 10 or more carbon atoms which may contain O, S, or N as a bonding atom to enhance the solubility of the cycloalkane-based solvent. n is an integer of 1 to 5. ]. When the hydrocarbon group having 10 or more carbon atoms enhances the solubility in the parent cycloalkane-based solvent, it has a branched chain and / or a substituent having a functional group that binds to the amino acid. Is. 6. The liquid phase peptide synthesis method according to claim 5, wherein the compound represented by the general formula A is selected from the following general formula group B. [Chemical 2] In each general formula, X, R and n are the same as in the general formula A.
Q is a single bond or a hydrocarbon group, R ₂ is a hydroxyl group, a thiol group, an amino group or a carbonyl group which binds to an amino acid, and R ₃ and R ₄ are groups of the following general formula C. [Chemical 3] R ₅ is a hydroxyl group, a thiol group, an amino group, or a carbonyl group which binds to an amino acid.