CN110590901A - Method for quickly constructing trace peptide library and synthesis device thereof - Google Patents
Method for quickly constructing trace peptide library and synthesis device thereof Download PDFInfo
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- CN110590901A CN110590901A CN201910964582.8A CN201910964582A CN110590901A CN 110590901 A CN110590901 A CN 110590901A CN 201910964582 A CN201910964582 A CN 201910964582A CN 110590901 A CN110590901 A CN 110590901A
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- 238000000034 method Methods 0.000 title claims abstract description 61
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 32
- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 32
- 108010067902 Peptide Library Proteins 0.000 title claims abstract description 22
- 238000006243 chemical reaction Methods 0.000 claims abstract description 111
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 65
- 238000005406 washing Methods 0.000 claims abstract description 49
- 150000001413 amino acids Chemical class 0.000 claims abstract description 46
- 238000005859 coupling reaction Methods 0.000 claims abstract description 46
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- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 31
- 238000010511 deprotection reaction Methods 0.000 claims abstract description 30
- 238000010168 coupling process Methods 0.000 claims abstract description 21
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- 238000010276 construction Methods 0.000 claims description 9
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- 238000010438 heat treatment Methods 0.000 description 1
- 229910000039 hydrogen halide Inorganic materials 0.000 description 1
- 239000012433 hydrogen halide Substances 0.000 description 1
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 1
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- OFIJLSUAGOAQOM-UHFFFAOYSA-N n,n-dimethylformamide;4-methylmorpholine Chemical compound CN(C)C=O.CN1CCOCC1 OFIJLSUAGOAQOM-UHFFFAOYSA-N 0.000 description 1
- ZUSSTQCWRDLYJA-UHFFFAOYSA-N n-hydroxy-5-norbornene-2,3-dicarboximide Chemical compound C1=CC2CC1C1C2C(=O)N(O)C1=O ZUSSTQCWRDLYJA-UHFFFAOYSA-N 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 230000004224 protection Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000011897 real-time detection Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000006277 sulfonation reaction Methods 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 1
- ITMCEJHCFYSIIV-UHFFFAOYSA-N triflic acid Chemical compound OS(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-N 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/10—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using coupling agents
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Structural Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a method for quickly constructing a trace peptide library and a synthesis device thereof, belonging to the field of polypeptide synthesis. The method solves the problems of low efficiency and the like of the existing SPOT method for constructing the peptide library. A method for quickly constructing a trace peptide library comprises the following steps: s01: derivatizing the surface of the lamellar solid phase carrier; s02: processing the lamellar solid phase carrier into a plurality of structural units; s03: filling the structural units into a reaction tank; s04: adding a coupling reaction solution into the reaction tank for coupling reaction, and directly adding a washing solvent to wash the reaction tank after the reaction; adding deprotection reaction liquid into the reaction tank to carry out deprotection reaction after washing is finished, and directly adding a washing solvent to wash the reaction tank after reaction; s05: repeating the step S04, coupling the amino acids one by one, coupling different amino acids, and selecting a coupling reaction solution corresponding to the amino acids to complete the loading of the fully-protected peptide chain on the lamellar solid phase carrier; the naked peptide or the loaded naked peptide is obtained after the cleavage. The invention has the advantages of high efficiency of constructing peptide library, and the like.
Description
Technical Field
The invention belongs to the field of polypeptide synthesis, and particularly relates to a method for quickly constructing a trace peptide library and a synthesis device thereof.
Background
The initial stage of research and development of new polypeptide drugs is mainly the discovery and structure optimization process of drug lead compounds. The weight requirement of the target polypeptide by the medicinal chemist in the process is not very large, but the sequence structure diversity of the target polypeptide is higher. Therefore, the rapid construction of a trace peptide library containing a large number of structural diversity members for corresponding pharmaceutical chemistry research is of great significance.
Constructing a peptide library by a traditional SPOT method, and starting with a carrier with a lamellar structure; the coupling and deprotection processes are realized by dripping reaction liquid on each planned site; thus, each amino acid synthesis building block is coupled to the carrier one by one, and finally, the whole peptide chain is constructed. The SPOT peptide library construction method is widely applied to relevant fields due to the characteristics of low cost, easy operation, convenient real-time detection and the like. However, the conventional SPOT method has some disadvantages:
firstly, the array sites on the surface of a specific sheet carrier are relatively dense, and the dropping operation of the reaction solution in the synthesis process needs to be careful so as to avoid influencing adjacent sites.
Secondly, the amount of the reaction solution in the polypeptide synthesis process cannot be increased, and only the reaction solution can be slowly diffused, otherwise, the adjacent sites are affected.
Moreover, washing of each site after reaction is troublesome, and the next round of washing can be performed only after the solution or solvent on the surface of the carrier is completely dried in the shade or air-dried, otherwise adjacent sites are affected.
In the process of constructing the peptide library by the traditional SPOT method, the synthesis efficiency is not high, the reaction process is time-consuming and labor-consuming, and the period is long due to the problems.
Disclosure of Invention
The first object of the present invention is to provide a method for rapidly constructing a library of peptides in minute quantities against the above-mentioned problems occurring in the prior art, and the second object of the present invention is to provide a synthesis apparatus for carrying out the above method.
The first object of the present invention can be achieved by the following technical solutions: a method for quickly constructing a trace peptide library is characterized by comprising the following steps:
s01: derivatizing the surface of the lamellar solid phase carrier;
s02: processing the lamellar solid phase carrier into a plurality of structural units;
s03: filling the structural units into a reaction tank;
s04: adding a coupling reaction solution into the reaction tank for coupling reaction, and directly adding a washing solvent to wash the reaction tank after the reaction; adding deprotection reaction liquid into the reaction tank to carry out deprotection reaction after washing is finished, and directly adding a washing solvent to wash the reaction tank after reaction;
s05: repeating the step S04, coupling the amino acids one by one, coupling different amino acids, and selecting a coupling reaction solution corresponding to the amino acids to complete the fully-protected peptide chain loaded by the lamellar solid phase carrier;
s06: and (4) cracking the fully-protected peptide chain loaded by the lamellar solid phase carrier obtained in the step (S05) by using a cracking solution to obtain naked peptide or naked peptide loaded by the lamellar solid phase carrier.
Preferably, when each building block is coupled to a different amino acid, each building block is located in a different reaction chamber.
Preferably, when a plurality of structural units need to be coupled with the same amino acid, the structural units needing to be coupled with the same amino acid can be marked with codes or filled into the same reaction tank in sequence, and the coupling reaction is carried out simultaneously.
Preferably, when the first coupled amino acid is the same, step S07, step S07: infiltrating coupling reaction liquid on the surface of the derivatized lamella solid phase carrier, and washing the surface of the derivatized lamella solid phase carrier by using a washing solvent after reaction; and soaking deprotection reaction liquid after washing to perform deprotection reaction, and washing with a washing solvent after the reaction to obtain the derivatized first amino acid-loaded lamella solid phase carrier.
Preferably, in step S01, the lamellar solid-phase carrier is one of a paper sheet, a porous polystyrene sheet, a glass sheet and a polyvinyl fluoride film.
Preferably, in step S01, the linking molecule for linking the first amino acid is: 4- [ (2, 4-dimethoxyphenyl) (Fmoc-amino) methyl ] phenoxyacetic acid; 3-hydroxy-9H-xanthen-9-one; 4-hydroxymethylphenylacetic acid; 4-benzyloxy benzyl alcohol, benzyl bromide or benzyl chloride.
Preferably, in step S02, the shape of the structural unit is one of circular, square, rectangular and regular triangle.
Preferably, the solvent in the coupling reaction solution is one or any combination of dichloromethane, trichloromethane, carbon tetrachloride, tetrahydrofuran, N' -dimethylformamide, 1, 4-dioxane and acetonitrile;
the condensation reagent in the coupling reaction liquid is N, N-diisopropyl carbodiimide, N, N-dicyclohexyl carbodiimide, 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride, 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethyl urea hexafluorophosphate/organic base, one or any combination of O-benzotriazole-tetramethylurea hexafluorophosphate/organic base, benzotriazole-1-oxytris (dimethylamino) phosphonium hexafluorophosphate/organic base, benzotriazole-1-oxytris (pyrrolidinophosphonium hexafluorophosphate/organic base, O-benzotriazole-N, N, N ', N' -tetramethylurea tetrafluoroborate/organic base;
the organic base is one or any combination of N, N-diisopropylethylamine, triethylamine and N-methylmorpholine;
the activating reagent in the coupling reaction liquid is one or any combination of N-hydroxysuccinimide, 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazol, 3-hydroxy-1, 2, 3-benzotriazin-4 (3H) -ketone or N-hydroxy-5-norbornene-2, 3-dicarboximide.
Preferably, the solvent in the deprotection reaction solution is one or any combination of dichloromethane, chloroform, carbon tetrachloride, tetrahydrofuran, N' -dimethylformamide, 1, 4-dioxane and acetonitrile;
the deprotection reagent in the deprotection reaction solution is one or any combination of trifluoroacetic acid (TFA), hydrogen chloride (HCl), hydrogen bromide (HBr), piperidine, diethylamine, triethylamine and 1, 8-diazabicyclo [5.4.0] undec-7-ene.
Preferably, the method for removing the side chain protecting group and breaking the chemical bond between the peptide chain and the linker is acidolysis reaction, saponification reaction or ammonolysis reaction, wherein the acidolysis solution in the acidolysis reaction is a mixed solution consisting of a, b and c:
a, one or any compound combination selected from formic acid, acetic acid, trifluoroacetic acid, trifluoromethanesulfonic acid and hydrogen halide;
b, one or any compound combination selected from the group consisting of methyl sulfide, anisole, ethanedithiol, triisopropylsilane, phenol and indole;
c, one or more mixed solvents selected from polar solvents;
the saponification liquid of the saponification reaction is a mixed solution consisting of d, e and f:
d is selected from NaOH, KOH, LiOH, CsOH, 1, 8-diazabicyclo (5.4.0) undec-7-ene, K2CO3、Cs2CO3One or any combination thereof;
e, water;
f, one or more mixed solvents selected from polar solvents;
the ammonolysis solution of the ammonolysis reaction is a mixed solution consisting of x, y and z:
x is selected from one or any combination of ethylamine, liquid ammonia, aliphatic ammonia and aromatic amine;
y, water;
z is one or more mixed solvents selected from polar solvents.
The second purpose of the invention is to provide a synthesis device for realizing the method, which is characterized by comprising a base, wherein the base is provided with a plurality of reaction tanks for containing carriers and reaction liquid, and the bottom of each reaction tank is provided with a liquid discharge hole.
Preferably, the carrier in the reaction tank completely covers the drain hole.
Preferably, the reaction tank further comprises a top cover for covering the reaction tank.
Preferably, the top cover is provided with a positioning retainer ring which is used for corresponding to each reaction tank opening one by one.
Preferably, the top cover is provided with a plurality of liquid feeding holes, and the liquid feeding holes are located in the center of the positioning retainer ring.
Preferably, the top cover is provided with a mark for marking the reaction tank.
Preferably, the diameter of the liquid adding hole is 5 mm.
Preferably, the diameter of the liquid discharge hole is 2 mm.
Preferably, the washing machine further comprises a liquid containing disc, the height of the liquid containing disc is lower than that of the base, and the liquid containing disc is used for containing washing liquid.
Preferably, the base and the top cover are both made of transparent materials.
The working principle of the invention is as follows: first, the surface of the lamellar solid support is derivatized. The carrier derivatization process is the process of linking the carrier and the linking molecule by covalent bonds.
The yield of the subsequent first amino acid coupling carrier is low due to the fact that functional groups carried on the surface of the lamellar solid phase carrier are dense, steric hindrance is large, the lamellar solid phase carrier is close to the carrier, activity is low and the like. To solve the above problem, a linker molecule (i.e., derivatization) is coupled to the surface of the carrier to facilitate attachment of the first amino acid.
Then, the lamellar solid phase carrier is physically processed into a plurality of structural units (for example, cut into a plurality of structural units by scissors), and filled into a reaction tank for coupling reaction. When coupling one by one, different coupling reaction solutions are selected according to different amino acids. The structural unit is the carrier in the reaction tank, i.e. the solid phase carrier of the sheet layer is cut into a plurality of small wafers, square wafers and the like by scissors, and one small wafer can be called as a structural unit. The shape of the structural unit is one of a circle, a square, a rectangle and a regular triangle.
After the reaction, the coupling reaction solution is not required to be air-dried or dried in the shade, a washing solvent can be directly added to wash the holes, after the washing is finished, next deprotection reaction is carried out, the deprotection reaction solution is added into the holes, after the reaction, the washing solvent can be directly added to wash the holes without waiting for the air-drying or the drying in the shade of the deprotection reaction solution, the step S04 is repeated, when the polypeptide is synthesized, different amino acids are required to be coupled, finally, side chain protecting groups are cut off, according to the property and the method of carrier modified connecting molecules, the free naked peptide or the naked peptide loaded by the lamella solid phase carrier in the covalent bond form can be obtained, and the free naked peptide or the naked peptide loaded by the lamella solid phase carrier in the.
One structural unit is used as a reaction site to synthesize one polypeptide. If identical amino acids are to be coupled, for example, the amino acids in the third position of the polypeptides to be synthesized are identical in the various structural units,
after the first amino acid is connected with the second amino acid, taking out each structural unit from the corresponding reaction tank, and then marking codes on each structural unit or stacking the structural units into the same reaction tank according to a specific sequence to carry out chemical reaction; and after the third amino acid is connected, each structural unit is arranged back to the corresponding reaction tank.
The use of condensing agents is divided into two cases: some condensation reagents can be directly added to promote the coupling reaction of amino acid; some of them must be added with a condensation agent and then added with a base to be effective. The organic bases mentioned above are this object.
Compared with the prior art, the invention has the following advantages:
1. compared with the traditional SPOT peptide library construction method, the method has the advantages that each site is effectively isolated by the reaction tank, and the feeding amount of the reaction reagent in the tank can be greatly increased, so that the reaction rate of each site can be greatly promoted, and the synthesis efficiency is improved. Secondly, the effective isolation of the sites can also avoid the cross contamination of adjacent sites during feeding, thereby greatly improving the feeding efficiency.
2. The washing process after coupling or deprotection at each site also has significant advantages over the traditional SPOT process. After the traditional SPOT method is used for washing each time, the next round of washing can be carried out only after the washing solvent on the surface of the carrier is completely dried in the shade or air-dried; in the invention, because each site is effectively isolated by the reaction tank, the sites in the tank can be directly washed for a plurality of times after reaction, and the whole process does not need air drying or shade drying, thereby greatly improving the washing efficiency of the post-treatment process.
3. In the traditional SPOT synthesis method, each site is fixed in the process of constructing a peptide library, and no matter how identical the loaded polypeptide sequence is, the connection of each amino acid in the loaded peptide sequence of each site must be completed one by one in the synthesis process. When a plurality of structural units need to be coupled with the same amino acid, the plurality of structural units can be filled into the same reaction tank to react simultaneously, and after the reaction is finished, the structural units are respectively reset, so that the 'mixed synthesis strategy' in combinatorial chemistry can be fully utilized. Thus, the synthesis efficiency is greatly improved.
4. Due to the advantages of 1-3, the method is greatly improved in the peptide library construction efficiency compared with the traditional SPOT method, is suitable for large-scale, rapid and effective high-flux construction of a trace lattice peptide library, and greatly improves the screening and structure optimization efficiency of the polypeptide drug lead compound.
Drawings
FIG. 1 is a Whatman 3MM CHR chromatography paper surface derivatization process of the present invention;
FIG. 2 is a synthesis of polypeptides of the invention using a derivatized Whatmann 3MM CHR vector.
FIG. 3 is a schematic view of the base of the present invention;
FIG. 4 is a schematic structural view of the top cover of the present invention;
FIG. 5 is a schematic structural view of a top cover label of the present invention;
FIG. 6 is a schematic structural view of a drip tray of the present invention;
FIG. 7 is an HPLC detection profile of the ZL-001 sequence of the present invention;
FIG. 8 is a MS detection map of the ZL-001 sequence of the present invention;
FIG. 9 is an HPLC detection profile of the ZL-002 sequence of the present invention;
FIG. 10 is an MS detection map of the present invention having the ZL-002 sequence;
FIG. 11 is an HPLC detection profile of the ZL-003 sequence of the present invention;
FIG. 12 is a MS detection map of the sequence numbered ZL-003 of the present invention.
In the figure, 1, a base; 2. a reaction tank; 3. a drain hole; 4. a top cover; 5. positioning a retainer ring; 6. a liquid adding hole; 7. identifying; 8. a liquid containing tray;
TFA: trifluoroacetic acid, DCM: dichloromethane, TsCl: p-toluenesulfonyl chloride, pyridine: pyridine, HBTU: o-benzotriazole-tetramethylurea hexafluorophosphate, DMF: n, N-dimethylformamide, NMM: n-methylmorpholine, 2, 6-dichorophenyl chloride: dichlorobenzoyl chloride, Ac2O: anhydrous acetic anhydride, solid phase peptide synthesis: solid phase polypeptide synthesis, clearage: cleavage, ethylamine: ethylamine, THF: tetrahydrofuran.
Detailed Description
The following are specific embodiments of the present invention and are further described with reference to the drawings, but the present invention is not limited to these embodiments.
The reagents and the lamellar solid phase carriers used in the examples of the present invention are commercially available.
The present invention uses Ala-Ile-X-Ile-X-Asn-Gln-Ala as template peptide sequence, and transforms the third and fifth sites (X) therein, and synthesizes the target peptide library with 20 polypeptides in Table 1.
TABLE 1 peptide library of interest
For the synthesis of the target peptide sequences in table 1, the synthesis blocks used are shown in table 2:
TABLE 2 synthetic building blocks
| Fmoc-Gly-OH | Fmoc-Arg(Pbf)-OH | Fmoc-Val-OH |
| Fmoc-Ala-OH | Fmoc-Asn(Trt)-OH | Fmoc-Lys(Boc)-OH |
| Fmoc-Ile-OH | Fmoc-Gln(Trt)-OH | Fmoc-Orn(Boc)-OH |
| Fmoc-Phe-OH | Fmoc-Pro-OH | Fmoc-Ser(tBu)-OH |
| Fmoc-Asp(OtBu)-OH | Fmoc-His(Trt)-OH | Fmoc-Trp(Boc)-OH |
| Fmoc-Glu(OtBu)-OH | Fmoc-Tyr(tBu)-OH | Fmoc-Leu-OH |
| Fmoc-Met-OH | Fmoc-Thr(tBu)-OH |
The synthetic building blocks of the present invention refer to the amino acids after various protections and derivatizations.
For example: if we need to introduce a Ser site into the polypeptide, we can obtain the target product through coupling with the corresponding peptide chain by using Fmoc-Ser (tBu) -OH as a synthetic block in the synthetic process and finally cutting. Dissolving the synthesized building block in proper solvent, adding condensation reagent and activating reagent to obtain the coupling reaction liquid.
As shown in FIG. 1-2, Whatman 3MM CHR paper (8 cm. times.12 cm) was selected as the sheet solid phase carrier (carrier 1).
The process I: derivatization and loading of the first amino acid on the surface of the support
a. Surface of chromatography paper p-toluene sulfonation treatment (preparation carrier 2)
Putting a piece of chromatographic paper into a 250ml conical flask, then adding 100ml of dichloromethane solution of 20% trifluoroacetic acid, and putting the conical flask on a shaking table to shake for 20 minutes; then pouring the solution in the bottle to dry, and washing the chromatographic paper in the bottle with DMF, methanol and dichloromethane each 100ml in sequence; and finally, taking out the chromatographic paper, and airing in the air.
Placing the dried chromatographic paper into a 250ml conical flask, adding a pyridine solution (100ml 2mol/L) of paratoluensulfonyl chloride (TsCl), placing the conical flask on a shaking table, shaking for 20 minutes, pouring out the solution in the conical flask, and washing the chromatographic paper in the conical flask by using DMF, methanol and dichloromethane which are 100ml respectively in sequence once; and finally, taking out the chromatographic paper, and airing in the air.
After the above process, a chromatography paper (support 2 in FIG. 1) having a surface p-toluenesulfonate was obtained.
b. PEG treatment of chromatography paper surface (preparation carrier 3)
Placing the chromatography paper (carrier 2) with the surface p-toluene sulfonated prepared in the process a into a 250ml conical flask; then adding 1mol/L of DMF solution of diethylene glycol bis (3-aminopropyl) ether; the liquid in the flask was then heated to 80 ℃ with a water bath. After heating for 40 minutes, cooling the liquid in the conical flask to room temperature; then washed with DMF, methanol, 2mol/L sodium hydroxide solution, water, methanol and dichloromethane each 100ml in sequence. And after washing, taking out the chromatographic paper, and airing in the air.
After the above process, the chromatography paper (carrier 3 in FIG. 1) with the surface being PEGylated was obtained.
c. Pam-PEG treatment of chromatography paper surface (preparation carrier 4)
The surface-PEGylated chromatography paper (support 3) prepared in Process b was placed in a 250ml Erlenmeyer flask, followed by the sequential addition of 4-hydroxymethylphenylacetic acid (670mg,4.0mmol), HBTU (1520mg,4.0mmol), DMF (100ml) and NMM (440. mu.L, 4.0mmol), followed by shaking the Erlenmeyer flask on a shaker for 20 minutes, followed by washing with 100ml each of DMF, methanol and dichloromethane.
HBTU is O-benzotriazole-tetramethyluronium hexafluorophosphate; DMF i.e. N, N-dimethylformamide; NMM is N-methylmorpholine.
After the above washing is completed, the chromatography paper is taken out and added into another 250ml conical flask, then 20% piperidine DMF solution is added for treatment for 20 minutes at room temperature, then the solution in the conical flask is poured out, and the conical flask is washed by 100ml each of DMF, methanol and dichloromethane for 1 time, finally the chromatography paper is taken out and dried in the air.
After the above procedure, a surface Wang-PEGylated chromatographic paper (Carrier 4 in FIG. 1) was obtained.
Pam-PEGylation refers to the treatment of PEGylation on the surface of a carrier and the modification of Pam Linker. The purpose is two:
1. after the surface of the carrier is subjected to PEGylation, the subsequent constructed peptide chain process can have a certain distance from the carrier, is not too close to the carrier, can be better solvated and is more beneficial to reaction.
2. Pegylation followed by pamylation: construction of peptide chains in the form of Pam linker is a common way of constructing peptide chains in polypeptide chemistry (in solid phase synthesis of polypeptides, accordingly, Pam resin is used). Using a polypeptide constructed by Pam linker, and obtaining a carrier-loaded naked peptide after the polypeptide is cracked by a lysate of which the main component is TFA; the naked peptide is obtained after cleavage by a cleavage solution comprising HF as the main component.
d. The surface of the chromatography paper is loaded with Fmoc-Ala structure (preparation carrier 5)
A250 ml Erlenmeyer flask was charged with the surface Wang-PEGylated chromatography paper (support 4) prepared in procedure c, then Fmoc-Ala-OH (1840mg,4.0mmol), DCM (100ml), 2, 6-dichlorobenzoyl chloride (1160. mu.L, 8.0mmol) and pyridine (1300. mu.L, 16mmol) were added sequentially, and the Erlenmeyer flask was shaken for 2 hours followed by washing with 100ml each 1 time sequentially with DMF, methanol, dichloromethane.
After the above washing, the chromatography paper was taken out and added into another 250ml conical flask, then 10% acetic anhydride and 6% NMM DMF solution were added, then the conical flask was put on a shaker and shaken at room temperature for 20 minutes, then the solution in the flask was poured out, and then each 100ml of DMF, methanol and dichloromethane was sequentially used for washing for 1 time, and finally the chromatography paper was taken out and air-dried.
After the above process, the chromatography paper (carrier 5 in figure 1) with Fmoc-Ala structure supported on the surface is obtained.
e. Chromatography paper surface loading deprotection treatment (preparation carrier 6)
The Fmoc-Ala-acylated CHR chromatography paper (support 5) with the surface prepared in process d was placed in a 250ml conical flask, then 100ml of 20% piperidine/DMF solution was added, then the conical flask was placed on a shaker and shaken at room temperature for 30 minutes, then the solution in the flask was poured out, followed by washing with DMF, methanol and dichloromethane in sequence, each 100ml for 1 time, and finally the chromatography paper was taken out and air-dried.
After the above process, the surface loaded NH is obtained2Chromatography paper of Ala structure (support 6 in FIG. 1). Thus, a chromatography paper loaded with the first amino acid Ala was obtained.
And (II) a process: polypeptide synthesis
a. Physical processing of the support
Process I the support 6 obtained in step e is processed into a wafer having a diameter of 1.5cm and placed in the corresponding reaction vessel of the synthesis apparatus.
b. Coupling side chain protected amino acids
According to the order of peptide synthesis to be performed in each well, the corresponding Fmoc protected amino acid (i.e., synthesis block) (0.4mmol), HBTU (0.152g,0.4mmol), N-methylmorpholine (44 ul) (0.4mmol) and 10ml DMF were selected, added to a 25ml Erlenmeyer flask, and then placed on a shaker, and shaken at room temperature for 15 minutes to prepare a coupling reaction solution. (several different amino acids are coupled, namely several different coupling reaction solutions are prepared; if the coupling amino acids needed at several sites are the same, one coupling reaction solution can be shared).
Adding the coupling reaction solution into corresponding reaction tanks one by one according to the volume of 200 ul/hole to completely wet Whatman 3MM CHR chromatographic paper in the reaction tanks, and standing for 30 minutes at room temperature; soaking a synthesis device in DMF, methanol and dichloromethane for 5 minutes respectively in sequence; and finally, air-drying in the air to obtain the chromatographic paper coupled with the second amino acid.
And (3) detecting the coupling degree of each site: add bromophenol blue in ethanol (0.01% w/v) to each well and allow to stand for 5 minutes. If the chromatography paper shows yellow, the coupling is complete; if blue color appears, the coupling is incomplete, and the amino acid at the site needs to be coupled repeatedly. After detection, the synthesis device is washed in an ethanol solvent and air-dried in the air for the next reaction.
c. Deprotection of chromatography paper loaded polypeptide
Preparing 20% piperidine solution in 200ml DMF as deprotection solution. Adding the deprotection solution into corresponding holes one by one according to the volume of 200 ul/hole to completely wet Whatman 3MM CHR chromatographic paper, and standing for 30 minutes at room temperature; then covering a glass cover, and soaking the specific SPOT glass device in DMF (dimethyl formamide), methanol and dichloromethane for 5 minutes respectively in sequence; and finally, air-drying in the air to obtain the chromatographic paper loaded with the deprotection peptide sequence.
d. Coupling one by one to complete the chromatography paper loading full protective peptide sequence (carrier 7)
Repeating the above processes b and c, coupling amino acids one by one, and loading the chromatography paper with fully-protected peptide chain (carrier 7, FIG. 2)
e. The side chain protecting group is cut off to obtain the chromatography paper loaded naked peptide (carrier 8)
Preparing 200ml of lysis solution, which comprises the following components: 95% trifluoroacetic acid/2.5% water/2.5% triisopropylsilane (vol/vol). According to the volume of 200 ul/hole, the cutting fluid is added into the corresponding holes one by one, so that Whatman 3MM CHR chromatographic paper in the cutting fluid is completely wetted, and the cutting fluid is kept stand for 30 minutes at room temperature; then covering a glass cover, and sequentially soaking the specific SPOT glass device in diethyl ether, methanol and dichloromethane for 5 minutes; finally, air drying in the air to obtain chromatography paper loaded naked peptide (carrier 8, figure 2)
The chromatography paper loaded naked peptide can be directly used for the activity screening of the polypeptide drug lead compound.
f. Carrier-loaded naked peptide structure and purity verification
To further verify the correctness of the sequence on the paper, we can use the method of ethaminolysis to detect the naked peptide after liberating, aiming at the Whatman 3MM CHR paper carrier loaded with polypeptide, as follows:
circular chromatographic papers of 1.5cm diameter were placed in separate test tubes, 1mL of 20% ethylamine in tetrahydrofuran was added, and the mixture was sealed and allowed to stand at room temperature for 4 hours. Then taking out the chromatographic paper; and concentrating the solution in the test tube to obtain an oil substance. And (3) taking a small amount of sample, dissolving with acetonitrile and water, and respectively detecting by RPHPLC and MS to obtain corresponding data. (see Table 3). As shown in FIGS. 7-12, due to the limitations of the patent space, the HPCL and MS maps for the accession numbers ZL-001, ZL-002, ZL-003 are only representatively shown in FIGS. 7-12, and the data obtained for the other accession numbers are shown in Table 3.
TABLE 3
As shown in FIGS. 3 to 6, a synthesis apparatus for rapidly constructing a library of peptides in minute quantities comprises a base 1, wherein the base 1 is provided with a plurality of reaction tanks 2 for accommodating a carrier and a reaction solution, and the bottom of the reaction tanks 2 is provided with drain holes 3.
More specifically, the carriers in the reaction tank 2 completely cover the drain holes 3. The carrier in the reaction tank 2 is a carrier processed into a sheet shape, and is called a sheet structure unit, and the sheet structure unit completely covers the liquid discharge hole 3, so that when the reaction liquid is dripped, the liquid does not leak from the liquid discharge hole 3 very quickly due to surface tension and the like, but slowly seeps out from the liquid discharge hole 3, and therefore the reaction liquid can stay in the reaction tank 2 for a long time and fully reacts with the functional group on the sheet structure unit.
Further, a top cover 4 for covering the reaction tank 2 is included. The top cover 4 is used for ensuring the accurate feeding position each time and avoiding the wrong feeding of the reaction liquid to an adjacent site; meanwhile, the dust collector can isolate external dust.
In further detail, the top cover 4 is provided with positioning collars 5 for one-to-one correspondence with the openings of each reaction tank 2. The top cover 4 is provided with a plurality of liquid feeding holes 6, and the liquid feeding holes 6 are positioned in the center of the positioning retainer ring 5. The positioning retainer rings 5 correspond to the openings of the reaction tanks 2 one by one, so that the liquid adding holes 6 correspond to the reaction tanks 2 one by one, and the reaction liquid can be accurately added into the corresponding reaction tanks 2 by adding the reaction liquid from the liquid adding holes 6.
More specifically, the top cover 4 is provided with a mark 7 for marking the reaction tank 2. The mark 7 is for marking the reaction tanks 2, because the reaction solution added to each reaction tank 2 may be different, if there is no mark, the operator is easy to add wrong reagents, for example, the reaction tank Aa needs to add a reagent, the reaction tank Ba needs to add B reagent, if there is no mark, the operator is easy to add B reagent to the reaction tank Aa and a reagent to the reaction tank Ba.
In more detail, the diameter of the liquid adding hole 6 is 5 mm.
More specifically, the diameter of the drain hole 3 is 2 mm.
In further detail, the washing machine also comprises a liquid containing disc 8, the height of the liquid containing disc 8 is lower than that of the base 1, and the liquid containing disc 8 is used for containing washing liquid. During washing, an operator can pour washing liquid into the liquid containing disc 8, then place the base 1 in the liquid containing disc 8, and the washing liquid enters the reaction tank 2 from the liquid discharge hole 3; the carrier in the reaction tank 2 can be sufficiently washed by moving the base 1 up and down.
In more detail, the base 1 and the top cover 4 are made of transparent materials. The transparent material is convenient for operators to observe and detect the progress of the coupling reaction and the deprotection reaction in the polypeptide synthesis.
After the surface of the lamellar solid phase carrier is derivatized, the lamellar solid phase carrier is processed into a plurality of lamellar structure units (namely, the carrier in the reaction tank 2) with proper sizes which can be placed in the reaction tank 2. The sheet layer solid phase carrier is one of paper sheet, porous polystyrene sheet, glass sheet and polyvinyl fluoride film. The carrier surface derivatization means that the surface of the carrier is treated with a chemical agent so as to load functional groups.
After the lamellar structure unit is placed into the reaction tank 2, the top cover 4 is covered, and then a liquid-transferring gun is used for adding prepared reaction liquid into the reaction tank 2 through the corresponding liquid-adding hole 6 on the top cover 4, so that the reaction liquid completely infiltrates the lamellar structure unit in the reaction tank 2. Along with the slow permeation of the reaction solution, the functional groups on the lamellar structure unit are fully converted to generate corresponding products; if the reaction is incomplete, the excess principle can be utilized to make the reaction completely converted by adding the reaction solution. After the reaction is finished, the whole device is placed into the liquid containing tray 8, and then a large amount of washing liquid is added for rinsing. This process is an amino acid coupling process with the polypeptide sequence loaded at each site.
After the completion of the washing, a previously prepared deprotection solution is added to each reaction vessel 2 in accordance with the above-described process of adding the reaction solution, and then a deprotection reaction is carried out. As the deprotection solution slowly permeates, the protecting group on the lamellar unit is sufficiently deprotected. After the reaction is finished, the whole device is placed into the liquid containing tray 8, and then a large amount of washing liquid is added for rinsing. The process is a deprotection process for loading polypeptide sequences at each site.
And (3) synthesizing a polypeptide sequence according to each site, and repeating the coupling and deprotection processes to complete the construction of the carrier loaded polypeptide sequence. If a plurality of lamellar structure units need to be connected with the same amino acid, the lamellar structure units can be added into the same reaction tank 2, and then the reaction solution is dripped, so that the same amino acid is connected to each structure unit. After the reaction is completed, the subsequent deprotection reaction is completed and the sheets are washed sufficiently, and then the sheet structure units are placed into the respective reaction tanks 2 again to perform the coupling of the respective next round of amino acid.
The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.
Although the terms base 1, reaction tank 2, drain hole 3, cover 4, positioning collar 5, filling hole 6, mark 7, drip tray 8, etc. are used more herein, the possibility of using other terms is not excluded. These terms are used merely to more conveniently describe and explain the nature of the present invention; they are to be construed as being without limitation to any additional limitations that may be imposed by the spirit of the present invention.
Claims (10)
1. A method for quickly constructing a trace peptide library is characterized by comprising the following steps:
s01: derivatizing the surface of the lamellar solid phase carrier;
s02: processing the lamellar solid phase carrier into a plurality of structural units;
s03: filling the structural units into a reaction tank (2);
s04: adding a coupling reaction solution into the reaction tank (2) for coupling reaction, and directly adding a washing solvent to wash the reaction tank (2) after the reaction; after washing is finished, adding deprotection reaction liquid into the reaction tank (2) for deprotection reaction, and directly adding a washing solvent to wash the reaction tank (2) after the reaction;
s05: repeating the step S04, coupling the amino acids one by one, coupling different amino acids, and selecting a coupling reaction solution corresponding to the amino acids to complete the loading of the fully-protected peptide chain on the lamellar solid phase carrier;
s06: and (4) cracking the fully-protected peptide chain loaded by the lamellar solid phase carrier obtained in the step (S05) by using a cracking solution to obtain naked peptide or naked peptide loaded by the solid phase carrier.
2. The method for rapid construction of peptide library according to claim 1, wherein each structural unit is located in a different reaction chamber (2) when each structural unit is coupled with a different amino acid.
3. The method for rapidly constructing a mini peptide library according to claim 1, wherein when a plurality of structural units need to be coupled with the same amino acid, the structural units needing to be coupled with the same amino acid can be labeled and coded or sequentially filled into the same reaction tank (2) to perform the coupling reaction simultaneously.
4. The method for rapidly constructing a mini peptide library according to claim 1, wherein when the first coupled amino acid of all the structural units is the same, the method further comprises step S07, step S07: infiltrating coupling reaction liquid on the surface of the derivatized lamella solid phase carrier, and washing the surface of the derivatized lamella solid phase carrier by using a washing solvent after reaction; and soaking deprotection reaction liquid after washing to perform deprotection reaction, and washing with a washing solvent after the reaction to obtain the derivatized first amino acid-loaded lamella solid phase carrier.
5. A synthesis device for implementing the method for rapid construction of a library of peptides as claimed in any one of claims 1 to 4, comprising a base (1), wherein said base (1) is provided with a plurality of reaction vessels (2) for containing carriers and reaction solutions, and the bottom of said reaction vessels (2) is provided with a drain hole (3).
6. A synthesis apparatus according to claim 5, characterized in that the support in the reaction vessel (2) completely covers the drainage holes (3), the diameter of the drainage holes (3) being 2 mm.
7. A synthesis device according to claim 5, characterized by further comprising a cover (4) for covering the reaction vessel (2), said cover (4) being provided with a marking (7) for marking the reaction vessel (2).
8. A synthesis unit according to claim 5, characterized in that the top cover (4) is provided with positioning collars (5) for one-to-one correspondence with the openings of each reaction vessel (2).
9. A synthesizer according to claim 5 characterised in that the top cover (4) is provided with a plurality of filling holes (6), the filling holes (6) being located centrally in the retaining ring (5), the filling holes (6) having a diameter of 5 mm.
10. A synthesis device according to claim 5, characterized by further comprising a liquid containing tray (8), wherein the height of the liquid containing tray (8) is lower than that of the base (1), the liquid containing tray (8) is used for containing washing liquid, and the base (1) and the top cover (4) are both made of transparent materials.
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| WO1991013084A1 (en) * | 1990-02-22 | 1991-09-05 | Boehringer Ingelheim Kg | Process and device for the simultaneous synthesis of several polypeptides |
| US5591646A (en) * | 1992-09-02 | 1997-01-07 | Arris Pharmaceutical | Method and apparatus for peptide synthesis and screening |
| CN103513039A (en) * | 2013-07-10 | 2014-01-15 | 广州美格生物科技有限公司 | Method used for detecting interaction of protein and other molecules by using directional peptide library |
| CN211057002U (en) * | 2019-10-11 | 2020-07-21 | 浙江昂拓莱司生物技术有限公司 | Synthesis device for rapidly constructing trace peptide library |
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2019
- 2019-10-11 CN CN201910964582.8A patent/CN110590901A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1991013084A1 (en) * | 1990-02-22 | 1991-09-05 | Boehringer Ingelheim Kg | Process and device for the simultaneous synthesis of several polypeptides |
| US5591646A (en) * | 1992-09-02 | 1997-01-07 | Arris Pharmaceutical | Method and apparatus for peptide synthesis and screening |
| CN103513039A (en) * | 2013-07-10 | 2014-01-15 | 广州美格生物科技有限公司 | Method used for detecting interaction of protein and other molecules by using directional peptide library |
| CN211057002U (en) * | 2019-10-11 | 2020-07-21 | 浙江昂拓莱司生物技术有限公司 | Synthesis device for rapidly constructing trace peptide library |
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