Polypeptide interlocking compound as well as preparation method and application thereof

Abstract

The invention discloses a method for efficiently preparing polypeptide interlocking compounds. The prepared polypeptide interlocking structure can improve the physical and chemical stability of polypeptide and enhance the efficacy of polypeptide molecules. The problems of tedious steps, low predictability and poor structural compatibility existing in a few strategies for synthesizing interlocking molecules with polypeptides as skeleton cores at present are solved. According to the invention, crown ether is utilized to activate amino groups in amino acid, and the crown ether and active amino-acid ester of another molecule are subjected to aminolysis reaction, so that the polypeptide interlocking compound can be prepared very efficiently.

Description

Technical Field

[0001] The invention belongs to the field of biosynthesis, and relates to a polypeptide interlocking structure system. The synthesis method includes both liquid phase synthesis and solid phase synthesis. Background Art

[0002] The drugability of peptide drugs is higher than that of general chemical drugs. They have high biological activity, strong specificity, relatively weak toxic reactions, are not easy to accumulate in the body, have relatively few interactions with other drugs, and have good affinity with receptors in the body. In view of this, many pharmaceutical companies have increased their research and development efforts on peptide drugs, especially in the fields of tumor and diabetes treatment. Many peptide drugs have been launched and have achieved huge economic benefits. Although peptide drugs have unique advantages, they also have disadvantages compared with small molecule chemical drugs: that is, the stability of peptide molecules is poor, they are easily degraded in the body, and thus have a short half-life. Continuous administration is required to maintain their efficacy, which brings inconvenience to patients. At present, after years of efforts by scientists, there are many methods that can effectively extend the half-life of peptide drugs in the body due to the easy degradation of peptide drugs. The first method is to change the structure of the peptide: design the polypeptide chain from the source, replace the amino acids that are easily cut by enzymes with other amino acids while ensuring the efficacy; or enhance the secondary structure of the polypeptide molecule to protect the cutting site, such as forming a cyclic peptide. The second method is to modify the polypeptide. The technology of modifying polypeptides with polyethylene glycol (PEG) is very mature internationally. Several protein polypeptide drugs have been successfully marketed after modification, such as PEG-modified interferon. The third method: polypeptides are combined with albumin [human serum albumin (HSA) or bovine serum albumin (BSA)], such as albumin and interleukin-2 fusion protein; polypeptides are coupled with exogenous or endogenous HSA, such as exenatide HSA to form recombinant albumin CJC-1134-PC, which has a half-life of 8 days after subcutaneous injection; if the molecular topological structure such as rotaxane is embedded in the polypeptide skeleton, the specific amino acid sequence in the polypeptide molecule can be protected without changing the polypeptide sequence. After protection, the spatial conformation of the amino acid sequence is changed and the spatial steric hindrance is increased, which may greatly improve the metabolic stability of the polypeptide molecule to the hydrolase at the amino acid site. Secondly, new structural modifications can be made to the molecular topological structural elements such as the crown ether ring to introduce new active sites to improve the physiological activity of the polypeptide. Summary of the invention

[0003] In order to overcome the defects of the prior art, the purpose of the present invention is to provide a method for efficiently preparing an interlocking polypeptide compound. In a solvent, compounds 1, 2, and 3 are added to a reaction bottle to react to obtain an interlocking polypeptide compound. The reaction route is:

[0004]

[0005] in,

[0006] R 1 = tert-butyloxycarbonyl or amino acid;

[0007] R 2 =iPr,Ph,-C 6 H 4 F, -C 6 H 4 Cl, -C 6 H 4 Br, -C 6 H 4 I, -C 6 H 4 OH, -C 6 H 4 SH, Me, Et, Pr, Bu, iBu, tBu, -CH 2 CH 2 SH, -CH 2 CH 2 SMe, -CONH 2 , -CH 2 CO 2 H, -CH 2 CH 2 CH 2 NH 2 , -OH, -SH, -C 3 H 3 N, -CH 2 CH 2 NHC(NH)NH 2 , -C 3 H 6 , -C 6 H 11 , -C 8 H 6 N;

[0008] X=-OC 6 H 5 NO 2 , -Cl, -Br;

[0009] R 3 =OMe, resin or amino acid;

[0010] R 4 =iPr,Ph,-C 6 H 4 F, -C 6 H 4 Cl, -C 6 H 4 Br, -C 6 H4 I, -C 6 H 4 OH, -C 6 H 4 SH, Me, Et, Pr, Bu, iBu, tBu, -CH 2 CH 2 SH, -CH 2 CH 2 SMe, -CONH 2 , -CH 2 CO 2 H, -CH 2 CH 2 CH 2 NH 2 , -OH, -SH, -C 3 H 3 N, -CH 2 CH 2 NHC(NH)NH 2 , -C 3 H 6 , -C 6 H 11 , -C 8 H 6 N;

[0011] Y = H;

[0012] Z = H;

[0013] Among them, amino acids include: aliphatic amino acids: alanine, valine, leucine, isoleucine, methionine, aspartic acid, glutamic acid, lysine, arginine, glycine, serine, threonine, cysteine, asparagine, glutamine, selenocysteine; aromatic amino acids: phenylalanine, tyrosine, tryptophan; heterocyclic amino acids: histidine, proline, pyrroletyrosine; heterocyclic imino acid: proline.

[0014] The crown ether ring (Compound 2) that can be used includes: 24 crown 8, benzo-24 crown 8, dibenzo-24 crown 8, naphtho-24 crown 8, anthracene-24 crown 8; 21 crown 7, benzo-21 crown 7, dibenzo-21 crown 7, naphtho-21 crown 7, anthracene-21 crown 7; 27 crown 9, benzo-27 crown 9, dibenzo-27 crown 9, naphtho-27 crown 9, anthracene-27 crown 9; 18 crown 6, benzo-18 crown 6, dibenzo-18 crown 6, naphtho-18 crown 6, anthracene-18 crown 6;

[0015] Wherein, the solvent is one or more of benzene and its derivatives, acetonitrile, alcohols, etc.; preferably, it is benzene.

[0016] Wherein, the molar ratio of compound 1, compound 2 and compound 3 is 2:1:2 or 1:1:1; preferably, it is 2:1:2.

[0017] Wherein, the reaction temperature is 25°C-100°C; preferably, it is room temperature.

[0018] Wherein, the reaction time is 2d-3d; preferably, 3d.

[0019] The method of the present invention can prepare all amino acid structures; it can be synthesized in a homogeneous system or obtained by solid phase synthesis in a heterogeneous system.

[0020] The innovation of the synthesis technology of the present invention lies in: utilizing crown ether and amino group activation to synthesize interlocked polypeptide compounds, which not only avoids complicated condition screening and substrate synthesis, but also achieves a substantial improvement in substrate and structural compatibility, has a high overall synthesis yield and is compatible with a variety of synthesis methods such as liquid phase and solid phase.

[0021] In a specific embodiment, the reaction steps are: using toluene as solvent, adding compounds 1, 2, and 3 into a reaction bottle in sequence, and reacting at room temperature for 3 days; after the reaction, adding silica gel to mix the sample and passing it through a column to obtain a white powder, which is a tripeptide interlocking compound.

[0022] The present invention constructs a natural amino acid polypeptide skeleton rotaxane for the first time through the strategy of crown ether stabilization of transition state. On this basis, a variety of polypeptide interlocking compounds are all new compounds prepared for the first time by the present invention. The design of synthetic axis primitive building blocks based on this strategy has the significant advantages of easy modification and functionalization. In addition, this simple strategy of mechanically interlocking peptides through macrocycles can also modify the biological activity of peptides by adjusting the molecular size, polarity, reactivity and stability of the peptides, and at the same time, it also lays the foundation for the design and synthesis of new polypeptide interlocking systems.

[0023] The present invention also provides a polypeptide interlocking compound, wherein the polypeptide chain is used as the axial unit and the crown ether is used as the cyclic unit, and the structural formula thereof is:

[0024]

[0025] R 1 = tert-butyloxycarbonyl or amino acid;

[0026] R 2 =iPr,Ph,-C 6 H 4 F, -C 6 H 4 Cl, -C 6 H 4 Br, -C 6 H4 I, -C 6 H 4 OH, -C 6 H 4 SH, Me, Et, Pr, Bu, iBu, tBu, -CH 2 CH 2 SH, -CH 2 CH 2 SMe, -CONH 2 , -CH 2 CO 2 H, -CH 2 CH 2 CH 2 NH 2 , -OH, -SH, -C 3 H 3 N, -CH 2 CH 2 NHC(NH)NH 2 , -C 3 H 6 , -C 6 H 11 , -C 8 H 6 N;

[0027] R 3 =OMe, resin or amino acid;

[0028] R 4 =iPr, Ph, -C 6 H 4 F, -C 6 H 4 Cl, -C 6 H 4 Br, -C 6 H 4 I, -C 6 H 4 OH, -C 6 H 4 SH, Me, Et, Pr, Bu, iBu, tBu, -CH 2 CH 2 SH, -CH 2 CH 2 SMe, -CONH 2 , -CH 2 CO 2 H, -CH 2 CH 2 CH 2 NH 2 , -OH, -SH, -C 3 H 3 N, -CH2 CH 2 NHC(NH)NH 2 , -C 3 H 6 , -C 6 H 11 , -C 8 H 6 N;

[0029] Y = H;

[0030] Z = H;

[0031] Among them, amino acids include: aliphatic amino acids: alanine, valine, leucine, isoleucine, methionine, aspartic acid, glutamic acid, lysine, arginine, glycine, serine, threonine, cysteine, asparagine, glutamine, selenocysteine; aromatic amino acids: phenylalanine, tyrosine, tryptophan; heterocyclic amino acids: histidine, proline, pyrroletyrosine; heterocyclic imino acid: proline.

[0032] The present invention also provides the use of the polypeptide interlocking compound in protecting biological active sites, transporting and delivering polypeptides, etc.

[0033] The beneficial effect of the present invention is that the present invention provides a method for efficiently preparing polypeptide interlocking compounds. Peptide interlocking structures are widely present in living organisms and have the effect of stabilizing polypeptide structures and improving the efficacy of polypeptides. However, there are very few methods for synthesizing polypeptide interlocking compounds reported previously, and the synthesis methods are complicated. The present invention utilizes the characteristic that crown ethers can activate amino groups in amino acids, and an aminolysis reaction with another molecule of active amino acid ester can be performed to prepare polypeptide interlocking compounds very efficiently. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the NMR image of the tripeptide interlocked compound 7.

[0035] Figure 2 This is the mass spectrum of tripeptide interlocked compound 7.

[0036] Figure 3 This is the NMR image of the tripeptide interlocked compound 10.

[0037] Figure 4 This is the mass spectrum of tripeptide interlocked compound 10.

[0038] Figure 5 This is the mass spectrum of the heptapeptide interlocked compound 11.

[0039] Figure 6 This is the enzymatic hydrolysis rate diagram of the heptapeptide interlocked compound 11. DETAILED DESCRIPTION

[0040] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0041] Example 1. Synthesis of interlocked polypeptide compound 7

[0042]

[0043] First, using toluene as solvent (10 ml), compounds 5 (77 mg, 0.2 mmol, 2.0 eq.), 2 (35 mg, 0.1 mmol, 1.0 eq.) and 6 (47 mg, 0.2 mmol, 2.0 eq.) were added into a reaction bottle in sequence and reacted at room temperature for 3 days. After the reaction, silica gel was added to mix the sample and passed through a column to obtain a white powder, which was the tripeptide rotaxane compound 7.

[0044] Example 2: Synthesis of interlocked polypeptide compound 10

[0045]

[0046] First, using toluene as solvent (10 ml), compounds 8 (70 mg, 0.2 mmol, 2.0 eq.), 2 (35 mg, 0.1 mmol, 1.0 eq.) and 9 (55 mg, 0.2 mmol, 2.0 eq.) were added into a reaction bottle in sequence and reacted at room temperature for 3 days. After the reaction, silica gel was added to mix the sample and passed through a column to obtain a white powder, which was the tripeptide rotaxane compound 10.

[0047] Example 3

[0048] By the synthesis method of Example 1 of the present invention, a [2]rotaxane with a heptapeptide skeleton was constructed with a good yield, and the structure is as follows:

[0049]

[0050] Secondary mass spectrometry analysis of rotaxanes revealed that positions without rotaxane protection were always broken first, while peptide bonds protected by rotaxanes remained stable. Enzymatic hydrolysis experiments on rotaxanes proved that their stability was enhanced. ( Figure 5 This is the mass spectrum of the heptapeptide interlocking compound 11. Figure 6 (Figure 11 is the enzymatic test diagram of the heptapeptide interlocking compound 11).

Claims

1. A polypeptide interlocking compound, characterized in that: The polypeptide chain is used as the axial unit and the crown ether is used as the ring unit. Its structural formula is: R1 = tert-butyloxycarbonyl or amino acid; R2=iPr, Ph, -C6H4F, -C6H4Cl, -C6H4Br, -C6H4I, -C6H4OH, -C6H4SH, Me, Et, Pr, Bu, <h2 style=";text-align:left;direction:ltr">iBu, tBu, -CH2CH2SH, -CH2CH2SMe, -CONH2, -CH2CO2H, -CH2CH2CH2NH2, -OH, -SH, -C3H3N, -CH2CH2NHC(NH)NH2, -C3H6, -C6H<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> ,-C8H6N; R3 = OMe, resin or amino acid; R4=iPr, Ph, -C6H4F, -C6H4Cl, -C6H4Br, -C6H4I, -C6H4OH, -C6H4SH, Me, Et, Pr, Bu, <h2 style=";text-align:left;direction:ltr">iBu, tBu, -CH2CH2SH, -CH2CH2SMe, -CONH2, -CH2CO2H, -CH2CH2CH2NH2, -OH, -SH, -C3H3N, -CH2CH2NHC(NH)NH2, -C3H6, -C6H<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> ,-C8H6N; Y = H; Z = H; Among them, amino acids include: aliphatic amino acids: alanine, valine, leucine, isoleucine, methionine, aspartic acid, glutamic acid, lysine, arginine, glycine, serine, threonine, cysteine, asparagine, glutamine, selenocysteine; aromatic amino acids: phenylalanine, tyrosine, tryptophan; heterocyclic amino acids: histidine, proline, pyrroletyrosine; heterocyclic imino acid: proline.

2. A method for preparing a polypeptide interlocking compound, characterized in that: In a solvent, compounds 1, 2, and 3 are added to a reaction flask for reaction to obtain a polypeptide rotaxane compound. The reaction route is as follows: Wherein, R1 = tert-butyloxycarbonyl or amino acid; R2=iPr,Ph,-C6H4F,-C6H4Cl,-C6H4Br,-C6H4I,-C6H4OH,-C6H4SH,Me,Et,Pr,Bu,iBu,tBu,-CH2CH2SH,-CH2CH2SMe,-CONH2,-CH2CO2H,-CH2CH2CH2NH2,-OH,-SH,-C3H3N,-CH2CH2NHC(NH)NH2,-C3H6,-C6H 11 ,-C8H6N; X = -OC6H5NO2, -Cl, -Br; R3 = OMe, resin or amino acid; R4=iPr, Ph, -C6H4F, -C6H4Cl, -C6H4Br, -C6H4I, -C6H4OH, -C6H4SH, Me, Et, Pr, Bu, <h2 style=";text-align:left;direction:ltr">iBu, tBu, -CH2CH2SH, -CH2CH2SMe, -CONH2, -CH2CO2H, -CH2CH2CH2NH2, -OH, -SH, -C3H3N, -CH2CH2NHC(NH)NH2, -C3H6, -C6H<h2 style=";text-align:left;direction:ltr"> 11 <h2 style=";text-align:left;direction:ltr"> ,-C8H6N; Y = H; Z = H; Among them, amino acids include: aliphatic amino acids: alanine, valine, leucine, isoleucine, methionine, aspartic acid, glutamic acid, lysine, arginine, glycine, serine, threonine, cysteine, asparagine, glutamine, selenocysteine; aromatic amino acids: phenylalanine, tyrosine, tryptophan; heterocyclic amino acids: histidine, proline, pyrroletyrosine; heterocyclic imino acid: proline.

3. The preparation method according to claim 2, characterized in that: The solvent is one or more of toluene and its derivatives, acetonitrile and alcohols.

4. The preparation method according to claim 2, characterized in that: The molar ratio of compound 1, compound 2 and compound 3 is 2:1:2 or 1:1:

1.

5. The preparation method according to claim 2, characterized in that: The reaction temperature is 25°C-100°C.

6. The preparation method according to claim 2, characterized in that: The reaction time is 2-3 days.

7. The polypeptide interlocking compound prepared by the method according to any one of claims 2 to 6.

8. Use of the polypeptide interlocking compound as claimed in claim 1 or 7 in protecting biological active sites, transporting and delivering polypeptides.

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