CN106854233B

CN106854233B - Quasi-peptide and preparation method and application thereof

Info

Publication number: CN106854233B
Application number: CN201710124106.6A
Authority: CN
Inventors: 赵子健; 朱凌; 高厚乾; 刘明珠; 杨延莲; 胡志远
Original assignee: Beijing Institute of Nanoenergy and Nanosystems
Current assignee: Beijing Institute of Nanoenergy and Nanosystems
Priority date: 2017-03-03
Filing date: 2017-03-03
Publication date: 2020-07-17
Anticipated expiration: 2037-03-03
Also published as: CN106854233A

Abstract

The invention provides a peptoid, a preparation method and an application thereof, wherein the peptoid comprises the following subunits of 1, 4-butanediamine (I), piperonyl amine (II), β -alanine (III), 1-naphthylamine (IV) and cysteine (V), the peptoid is simple to synthesize and has stronger binding capacity with α -synuclein, and can effectively screen PD patients and normal human serum through α -synuclein in serum, thereby providing a new liquid biopsy method and thought for diagnosing and monitoring Parkinson's disease.

Description

Quasi-peptide and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicine and pharmacology, and relates to a peptide, and a preparation method and application thereof.

Background

Parkinson's Disease (PD) is a very common degenerative Disease of the nervous system, with an average age of around 60 years in the high-and advanced-age population. Until now, the prevalence of PD in people over 65 years of age in our country has reached 1.7% (although this figure may be artificially low, since many patients in rural areas have never been diagnosed), and most parkinson's disease patients are sporadic cases, with less than 10% of patients having a comparable family genetic history. The most important pathological change of the Parkinson's disease is degeneration and death of midbrain nigral Dopamine (DA) neurons, and thus the striatal DA content is remarkably reduced to cause diseases. However, despite the numerous studies on Parkinson's disease, the precise etiology is still unclear, and genetic factors, environmental factors, aging, oxidative stress, and the like may be involved in the degenerative death process of PD dopaminergic neurons. The disease was first described in detail by James parkinsonon in 1817, who had clinical manifestations mainly including resting tremor, bradykinesia, myotonia and postural gait disorders, with patients possibly associated with non-motor symptoms such as depression, constipation and sleep disorders. The diagnosis of Parkinson's disease depends primarily on medical history, clinical symptoms and signs. The general auxiliary inspection is mostly free from abnormal changes. Drug therapy is the most prominent treatment for parkinson's disease. Levodopa formulations remain the most effective drug. Surgical treatment is an effective supplement to medical treatment. Rehabilitation therapy, psychological therapy and good care can also improve symptoms to a certain extent. Although the currently applied treatment means can only improve symptoms, cannot prevent the progress of the disease and cannot cure the disease, the effective treatment can significantly improve the life quality of patients. The life expectancy of PD patients is not significantly different from the general population. However, as China is the first major country of the global population, the current aging social phenomenon is more and more serious, so that the Parkinson's disease becomes a major problem which must be faced by the China society and medical treatment.

The prominent pathological changes in Parkinson's disease are degenerative death of mesolimbic Dopaminergic (DA) neurons, a significant reduction in striatal DA content, and the appearance of eosinophilic inclusions within the cytoplasm of the substantia nigra residual neurons, i.e., Lewy bodies (L ewy bodies), clinical symptoms of at least greater than 50% of the nigral dopaminergic neurons dying and greater than 80% of the striatal DA content, in addition to the dopaminergic system, significant impairment of the non-dopaminergic system of Parkinson's disease patients, cholinergic neurons such as the Meynert basal ganglia, noradrenergic neurons of the locus ceruleus, 5-hydroxytryptamine neurons of the nucleus pulposus of the brain stem, and neurons of the cerebral, brain stem, spinal cord, and peripheral autonomic nervous system, the significant reduction in striatal dopamine content is closely associated with the appearance of motor symptoms of Parkinson's disease, the significant reduction in the mesolimbic and mesocortical dopamine concentrations is closely associated with the appearance of intelligent decline, affective disorders, etc. α -synuclein is a protein closely associated with the peripheral synaptic nervous system and its peripheral synaptic protein, a major synaptic dysfunction and pathogenesis of Parkinson's disease.

It has been found that α -synuclein is in a dynamic equilibrium between normal, misfolded and oligomerized state, and when this equilibrium is broken, fibrils aggregate rapidly into macromolecules and insoluble fibrils, α -synuclein exhibits many forms under different influences, including an extended state, a pre-lytic globular state, α -helical state (membrane bound), β -lamellar state, dimeric state, oligomeric state, and insoluble amorphous and fibrous state, structural changes due to point mutations in α -synuclein, increased intracellular content, accumulation of large amounts of protein molecules, structural sequence truncation, intracellular anion and salt concentrations (changes in pH), neurotoxic molecules (heavy metals, organic solvents, carbon monoxide, MPTP, pesticides and herbicides), post-translational modifications (oxidation, phosphorylation and nitration), and the like, all promote α -synuclein to aggregate into poorly soluble fibers, and α -synuclein in the oligomeric state is currently considered to be the most cytotoxic.

According to the characteristics of insidious onset and gradual progress, unilateral involvement is involved and further developed to the contralateral side, the symptoms are static tremor and slow movement, and clinical diagnosis can be made by eliminating atypical parkinsonism-like symptoms.

Since it was discovered in 1997 that α -synuclein was involved in familial Parkinson's disease gene (SCNA) mutations and major components of Lewy bodies, it became the focus in molecular pathogenesis in Parkinson's disease, and we invented a peptide-like peptide to detect α -synuclein content in blood in a simpler, more sensitive, less costly, non-invasive method to diagnose and monitor PD. -like peptide (peptoid) is a non-natural foldoid with N-substituted glycine as a unit, similar to polypeptide structure, that can fold into a highly bioactive and highly specific functional unit, and that is more abundant in its constitutional units than polypeptides, and is resistant to proteases, and thus, the peptide-like compounds have good biological and chemical properties.

Therefore, the development of an agent capable of being used for parkinson's disease detection, diagnosis and/or monitoring in the art is a problem to be solved in the art.

Disclosure of Invention

The peptoid is simple to synthesize, has strong binding capacity with α -synuclein, and can effectively screen PD patients and normal human serum through α -synuclein in the serum.

In order to achieve the purpose, the invention adopts the following technical scheme:

in one aspect, the invention provides a peptoid comprising the subunits 1, 4-butanediamine (I), piperonylamine (II), β -alanine (III), 1-naphthylamine (IV), and cysteine (V).

In the present invention, the structure of the donor of each subunit of the peptoid is as follows:

preferably, the sequence of the peptoid subunits is I-IV-III-II-I-I-V.

Preferably, in the present invention, the peptoid has the following structure:

in the invention, the peptoid with the structure has stronger binding capacity with α -synuclein, and can effectively screen the serums of Parkinson disease patients and normal people through α -synuclein in the serums.

In a second aspect, the present invention provides a method of preparing a peptoid as described above, the method of preparation being synthesized by solid phase synthesis.

Preferably, the preparation method comprises the following steps:

(1) attaching a first subunit of a peptoid to a solid support in order of attachment of subunits of the peptoid;

(2) reacting bromoacetic acid with an amino group of a first subunit attached to a solid support under activation of an activator to form an amide bond;

(3) reacting a donor of a second subunit of the peptoid with the product obtained in the step (2) to replace bromine atoms, thereby completing the connection of the second subunit;

(4) then bromoacetic acid and the connection of subsequent subunits are repeatedly carried out until the connection of all subunits is completed;

(5) and (3) cleaving the synthesized peptoid from the solid phase carrier to obtain the peptoid.

Preferably, the solid phase carrier in the step (1) is Rink amide AM resin.

Preferably, the solid support is swollen prior to attaching the first subunit of the peptoid to the solid support in step (1).

When the solid phase carrier is Rink amide AM resin, the solid phase carrier is swelled and deprotected by piperidine to expose the amino group of Rinkamide AM resin.

Preferably, the attachment of the first subunit of the peptoid to the solid support is performed under the action of a condensing agent and an activating agent.

Preferably, the condensing agent is any one or a combination of at least two of 2- (3' -N-oxo-benzotriazole) -1,1',3,3' -tetramethylurea hexafluorophosphate, O-benzotriazole-N, N, N ', N ' -tetramethylurea tetrafluoroborate and 1-hydroxybenzotriazole.

Preferably, the activator is N-methylmorpholine.

Preferably, the activating agent in step (2) is N, N' -diisopropylcarbodiimide or dicyclohexylcarbodiimide.

Preferably, the temperature of the reaction in step (2) is 20-40 ℃, such as 20 ℃, 22 ℃, 25 ℃, 28 ℃, 30 ℃, 32 ℃, 35 ℃, 37 ℃, 39 ℃ or 40 ℃.

Preferably, the reaction time in step (2) is 20-100min, such as 20min, 25min, 30min, 35min, 40min, 50min, 60min, 70min, 80min, 90min or 100 min.

In step (3) the donor is a compound capable of providing the peptoid subunit, e.g., the donor for the cysteine subunit is cysteine, the donor for the β -alanine subunit is β -alanine, and so on.

Preferably, the temperature of the reaction in step (3) is 20-40 ℃, such as 20 ℃, 22 ℃, 25 ℃, 28 ℃, 30 ℃, 32 ℃, 35 ℃, 37 ℃, 39 ℃ or 40 ℃.

Preferably, the reaction time in step (3) is 50-150min, such as 50min, 55min, 60min, 65min, 70min, 80min, 90min, 100min, 120min, 140min or 150 min.

In the present invention, the bromoacetic acid and the subsequent subunit ligation are repeated in step (4), i.e., steps (2) and (3) are repeated, except that the subunit ligated is a subsequent subunit.

Preferably, the cracking agent used in the cracking in the step (5) comprises the following components in percentage by mass: 95% trifluoroacetic acid, 2.5% ultrapure water and 2.5% triisopropylsilane.

Preferably, groups which do not participate in the linking reaction may be protected during the preparation of the polypeptide subunit to ensure the accuracy of the linking site, so that the reaction proceeds more accurately and smoothly, and then deprotection is performed to remove the protecting groups after the linking of all subunits is completed.

In another aspect, the invention provides an amyloid detection agent comprising a peptoid as described above.

The peptoid of the invention can be used as a detection agent for detecting amyloid protein or a component of the detection agent for detecting amyloid protein.

Preferably, the amyloid protein is α -synuclein.

In another aspect, the present invention provides a pharmaceutical composition comprising a peptoid as described above.

Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

Preferably, the pharmaceutically acceptable adjuvant is any one or a combination of at least two of excipient, diluent, carrier, flavoring agent, binder or filler.

In another aspect, the present invention provides the use of a peptoid or a pharmaceutical composition as described above for the preparation of a medicament for the detection, diagnosis and/or monitoring of diseases related to α -synuclein.

Preferably, the disease is parkinson's disease.

Compared with the prior art, the invention has the following beneficial effects:

(1) the binding capacity of the peptoid of the invention and α -synuclein is stronger, and the equilibrium dissociation constant KD in the binding kinetic constant of the peptoid of the invention and α -synuclein, which is obtained by the surface plasmon resonance technology, is 10^-8On the order of moles/liter;

(2) the contrast of the blood signal intensity of PD patients and normal persons by detecting the peptoid by using the surface plasmon resonance technology can find that the patients and the normal persons can be obviously identified by using the peptoid;

(3) the PD diagnosis technology based on the peptide can realize noninvasive label-free rapid diagnosis;

(4) the peptide-like peptide of the invention has simple synthesis, high efficiency and low cost.

Drawings

FIG. 1 is a graph of the results of resonance assays for surface plasmonics bound by the peptoid molecules of the present invention with α -synuclein concentrations of 2.632 μ M, 1.316 μ M, 0.658 μ M, 0.329 μ M;

FIG. 2 is a graph showing the results of the test for specific binding of the peptoids of the present invention to α -synuclein;

FIG. 3 is a graph showing the effect of the peptoids of the present invention on the serum signals of PD patients compared with normal persons;

FIG. 4 is a graph showing the results of sensitivity tests for detecting serum in a PD diagnostic system using the peptoid of the invention.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Example 1

In this example, peptoids were synthesized by solid phase subunit synthesis, specifically including the following steps:

(1) rink amide AM resin (substitution level 0.3mmol/g) was swollen and deprotected with piperidine, cysteine was equimolar mixed with 2- (3' -N-oxo-benzotriazole) -1,1',3,3' -tetramethyluronium hexafluorophosphate and coupled under activation of N-methylmorpholine.

(2) Adding 2M bromoacetic acid and 3.2M N, N' -Diisopropylcarbodiimide (DIC) into Rink amide AM resin, reacting at 37 deg.C for 30min, and acylating the amino group at the end of the resin;

(3) adding 2M primary amine to react for 90min at 37 ℃, and replacing bromine atoms through nucleophilic substitution reaction to complete the synthesis of a subunit;

(4) repeating the steps (2) and (3) until the synthesis of the rest units is completed;

(5) after the synthesis was complete, the side chain protecting groups were removed and the peptoid cleaved from the resin with 95% trifluoroacetic acid, 2.5% ultrapure water, 2.5% triisopropylsilane for future use.

The peptoids prepared in this example have the following formula:

example 2

(1) rink amide AM resin (substitution level 0.3mmol/g) is swelled and deprotected by piperidine, cysteine and O-benzotriazole-N, N, N ', N' -tetramethylurea tetrafluoroborate are mixed in equimolar, and coupling is carried out under the activation of N-methylmorpholine.

(2) Adding 2M bromoacetic acid and 3.2M N, N' -Diisopropylcarbodiimide (DIC) into Rink amide AM resin, reacting at 25 deg.C for 60min, and acylating the amino group at the end of the resin;

(3) adding 2M primary amine to react for 50min at 25 ℃, and replacing bromine atoms through nucleophilic substitution reaction to complete the synthesis of a subunit;

(5) after the synthesis was complete, the side chain protecting groups were removed and the peptoid cleaved from the resin with 95% trifluoroacetic acid, 2.5% ultrapure water, 2.5% triisopropylsilane for future use. The synthetic peptoids have the molecular structure described in example 1.

Example 3

(1) rink amide AM resin (substitution level 0.3mmol/g) was swollen and deprotected with piperidine, cysteine was equimolar mixed with 1-hydroxybenzotriazole and coupled under activation of N-methylmorpholine.

(2) Adding 2M bromoacetic acid and 3.2M dicyclohexylcarbodiimide into Rink amide AM resin, reacting for 20min at 40 ℃, and acylating the amino group at the tail end of the resin;

(3) adding 2M primary amine to react for 150min at 20 ℃, and replacing bromine atoms through nucleophilic substitution reaction to complete the synthesis of a subunit;

Example 4

In this example, the binding ability between the peptoid and α -synuclein was examined as follows:

the specific steps for testing the binding capacity between the peptoid and the α -synuclein by using the surface plasmon resonance imaging technology are as follows:

(1) solubilization of peptoids into ddH₂In O, the concentration is 1-1000 MuM;

(2) samples were spotted onto the surface of an SPRi chip, 3 spots for each sample were repeated, and after incubation at 4 ℃ for 12 hours, the samples were washed dry with 10 XPBS, 1 XPBS, and ultrapure water. Then sealing the chip with 1M aminoethanol hydrochloride for 30 minutes, then cleaning with ultrapure water for 5 times, and finally drying with clean nitrogen;

(3) mounting the chip on an SPRi instrument, measuring an SPRi angle, adjusting to an optimal optical position, selecting related detection points in a detection area, wherein the related detection points comprise a sample point and a blank point, and setting the experiment flow rate to be 2 mu L/s;

(4) PBS is selected as a buffer solution and is introduced into a flow cell until a base line is stable, then detection is sequentially carried out by the concentration of 2.632 mu M, 1.316 mu M, 0.658 mu M and 0.329 mu M, the combination time is 300 seconds, the dissociation time is 300 seconds, and phosphoric acid is introduced into each concentration for regeneration.

The detection result is shown in FIG. 1 (wherein △ AU is used for reflecting binding in surface plasmon resonance imagingUnit of signal intensity, a dimensionless unit) as shown, fitted, and balanced for dissociation constant K_DIs 6.18 × 10^-8Mols/liter, indicating that the peptoid has a relatively high level of affinity for α -synuclein.

Example 5

In this example, peptoids were tested for specificity for α -synuclein binding as follows:

the surface plasmon resonance imaging technology is used for testing the specificity of the binding α -synuclein by the peptoid, and the specific steps are as follows:

(1) the same SPRi chip as in example 4 was fabricated, mounted on an SPRi instrument, the SPRi angle was measured and adjusted to the optimal optical position, and the relevant detection points including the sample point and the blank point were selected in the detection area, and the experimental flow rate was set to 2. mu. L/s;

(2) PBS is selected as buffer solution and is introduced into a flow cell until a base line is stable, and then binding tests are sequentially carried out on α -synuclein (a-syn), Human Serum Albumin (HSA), IgG, Transferrin (TRF) and IgM with the concentration of 2.632 mu M, the binding time is 300 seconds, the dissociation time is 300 seconds, and phosphoric acid is introduced into each concentration for regeneration.

As shown in FIG. 2, the binding of the peptoid of the present invention to α -synuclein has high specificity.

Example 6

In this example, the serum signal was detected using a peptoid as follows:

the method for detecting the serum of PD patients and the normal human serum by using the surface plasma resonance imaging technology comprises the following specific steps

(2) PBS is selected as a buffer solution to be filled into a flow cell until a baseline is stable, serum diluent (1:5000) of different patients and normal people is respectively filled into the flow cell, the dissociation time is 300 seconds, and phosphoric acid and proteinase K are filled into each sample for regeneration.

The results are shown in FIG. 3, which are lg values (i.e., lgC) at different peptoid concentrations_PDI2) The signal binding strength of the bottom surface plasmon resonance imaging is shown in fig. 3, and when the concentration of the peptoid is higher, the PD patient can be clearly distinguished from the normal person (normal).

Example 7

In this example, the sensitivity of the PD diagnostic system was tested using peptoids as follows:

the surface plasma resonance imaging technology is utilized to test the sensitivity of the PD diagnostic system for detecting serum, and the specific steps are as follows:

(2) PBS is selected as a buffer solution to be filled into a flow cell until a baseline is stable, serum diluent of different patients and normal people is respectively filled, the dilution concentration is respectively 1:2000, 1:4000, 1:8000, 1:16000 and 1:32000, the combination time is 300 seconds, the dissociation time is 300 seconds, and phosphoric acid and proteinase K are filled into each sample for regeneration.

The detection result is shown in figure 4, when the serum dilution ratio is less than or equal to 1:8000, the PD patient can be obviously distinguished from the normal person, and the extremely high sensitivity is demonstrated.

In conclusion, the peptoid is a peptoid which can be combined with α -synuclein in serum with high sensitivity and high specificity, and provides a novel liquid biopsy method and an idea for diagnosing and monitoring Parkinson's disease.

The applicant states that the invention is illustrated by the above examples to the peptoids of the invention and the methods of preparation and use thereof, but the invention is not limited to the above examples, i.e. it is not meant to imply that the invention must be practiced in the above examples. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.

Claims

1. A peptoid comprising the subunits 1, 4-butanediamine (I), piperonylamine (II), β -alanine (III), 1-naphthylamine (IV) and cysteine (V),

the sequence of the subunit of the peptoid is I-IV-III-II-I-I-V;

the peptoid has the structure:

2. the method of claim 1, wherein the peptoid is synthesized by solid phase synthesis.

3. The method of manufacturing according to claim 2, comprising the steps of:

4. The method according to claim 3, wherein the solid phase carrier in the step (1) is Rink aminodeeAM resin.

5. The method of claim 3 or 4, wherein the solid support is swollen prior to the step (1) of attaching the first subunit of the peptoid to the solid support;

6. The method of claim 5, wherein the step of attaching the first subunit of the peptoid to the solid support is performed by the action of a condensing agent and an activating agent.

7. The method according to claim 6, wherein the condensing agent is any one or a combination of at least two of 2- (3' -N-oxo-benzotriazole) -1,1',3,3' -tetramethylurea hexafluorophosphate, O-benzotriazole-N, N, N ', N ' -tetramethylurea tetrafluoroborate and 1-hydroxybenzotriazole.

8. The method of claim 6, wherein the activator is N-methylmorpholine.

9. The production method according to claim 3, wherein the activating agent in the step (2) is N, N' -diisopropylcarbodiimide or dicyclohexylcarbodiimide.

10. The method according to claim 3, wherein the temperature of the reaction in the step (2) is 20 to 40 ℃.

11. The method according to claim 3, wherein the reaction time in the step (2) is 20 to 100 min.

12. The method according to claim 3, wherein the temperature of the reaction in the step (3) is 20 to 40 ℃.

13. The method according to claim 3, wherein the reaction time in the step (3) is 50 to 150 min.

14. The preparation method according to claim 3, wherein the cracking agent used in the cracking in the step (5) comprises the following components in percentage by mass: 95% trifluoroacetic acid, 2.5% ultrapure water and 2.5% triisopropylsilane.

15. The method according to claim 3, wherein a group not involved in the ligation reaction is protected during the ligation of the subunits of the peptoid, and then deprotected to remove the protecting group after the ligation of all the subunits is completed.

16. An amyloid assay agent comprising the peptoid of claim 1, wherein the amyloid is α -synuclein.

17. A pharmaceutical composition comprising the peptoid of claim 1.

18. The pharmaceutical composition of claim 17, further comprising a pharmaceutically acceptable excipient.

19. The pharmaceutical composition of claim 18, wherein the pharmaceutically acceptable excipient is any one or a combination of at least two of an excipient, a diluent, a carrier, a flavoring agent, a binder, or a filler.

20. Use of the peptoid of claim 1 or the pharmaceutical composition of claim 17 for the preparation of a medicament to detect, diagnose and/or monitor α -synuclein-associated diseases, which is parkinson's disease.