CN112679574B - Polypeptide disulfide bond synthesis method based on penicillamine and application thereof - Google Patents
Polypeptide disulfide bond synthesis method based on penicillamine and application thereof Download PDFInfo
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Abstract
The invention provides a polypeptide disulfide bond synthesis method based on penicillamine, which adopts a route 1 or 2, wherein the route 1 is as follows: dissolving iodine in N, N-dimethylformamide, adding into the synthesized resin A with related sequence, blowing with nitrogen gas for reaction, cutting off polypeptide with shearing liquid, and separating and purifying with high performance liquid chromatography to obtain target product; route 2 is: cutting the polypeptide of the synthesized related sequence from the resin A by using a shearing liquid, separating the polypeptide of unoxidized cyclization by using a high performance liquid chromatography, dissolving the polypeptide in a phosphate buffer solution with the pH value of 7.4-8.0, adding an oxidant for oxidation, and separating and purifying by using the high performance liquid chromatography to obtain a target product; the invention provides a design and synthesis strategy of a stable disulfide bond and applies the disulfide bond as an effective covalent reaction warhead in the field of chemical biology.
Description
Technical Field
The invention belongs to the field of stable polypeptide methodology and design and development of chemical biological polypeptide inhibitors, and particularly relates to a polypeptide disulfide bond synthesis method based on penicillamine and application thereof, in particular to a method for designing and synthesizing a stable disulfide bond and exploring the application of the stable disulfide bond in the field of chemical biological research by taking the development of a polypeptide covalent inhibitor as an example.
Background
The polypeptide drug is between the traditional small molecule drug (< 500 Da) and the biological macromolecule drug (> 5000 Da), combines the advantages of the two drugs, such as better affinity, specificity, biocompatibility, safety and the like, can effectively target Protein and Protein Interaction (PPI) interfaces which are difficult to target by other drugs, and fills corresponding gaps. Polypeptide drugs have also gained increased attention in recent years. To date, over 60 polypeptide drugs have been used for disease treatment, and over 140 are in clinical research. However, the linear polypeptide is easy to degrade in vivo, has poor membrane penetration capability, low oral bioavailability and the like, so that further biological application of the linear polypeptide is limited. Researchers have attempted to modify polypeptides by various chemical means in order to obtain polypeptides with better stability, stronger cell penetration ability, and certain secondary conformation, thereby expanding the application range of polypeptides.
In these chemical modification methods, side chain stabilization and N-terminal induced nucleation are the main factors. However, many of the methods developed today increase the difficulty of synthesizing modified polypeptides to a greater or lesser extent, including the introduction of too many unnatural amino acids, the use of more complex chemical reactions, and the introduction of too many additional linkers, such as benzyl, tetrafluoro-substituted benzenes, etc. Among these, disulfide bonds that do not rely on too many unnatural amino acids and complex chemical reactions and too many extrinsic group modifications have attracted considerable attention. Disulfide bonds play important roles in biological functions as a specific structure of native polypeptides and protein conformational constraints. However, the use and development of disulfide bonds has been limited due to the stability of disulfide bonds themselves and the difficulty of correct recombination of disulfide bonds in native disulfide-rich polypeptides or proteins. The study of the disulfide bond formation from Penicillamine (Penicillamine) originated in the eighties of the last century brought a little hope for this limitation. It was found that the disulfide bonds formed between penicillamine and cysteine, and also on penicillamine itself, are much more stable than the disulfide bonds formed between cysteines.
Based on the research results, the invention is urgently needed to introduce the disulfide bond into the modification of the stabilized polypeptide, and simultaneously, the disulfide bond can be skillfully applied to the chemical biological research of reconstructing a new disulfide bond near cysteine, so that the biological functions of the novel disulfide bond can be further expanded, and more subsequent biological applications are provided with ideas.
Disclosure of Invention
In view of the above technical problems in the prior art, the present invention provides a method for synthesizing a polypeptide disulfide bond based on penicillamine, and more particularly, a method for stabilizing a polypeptide by forming a disulfide bond between penicillamine and cysteine and also penicillamine itself and applying the same to chemical and biological studies near driving the reconstruction of disulfide bonds. The method creatively develops the application of the disulfide bond and also expands the types of warheads in the targeted induction of covalency.
The invention provides a polypeptide disulfide bond synthesis method based on penicillamine, the structural general formula of the polypeptide disulfide bond is shown as the formula (I),
c is cysteine or penicillamine, n is 2 or 3, AA represents amino acid without sulfydryl except penicillamine and cysteine, pen is penicillamine, the synthesis method adopts route 1 or route 2,
wherein R of the resin A 1 And R 2 Is a mercapto protecting group selected from one of trityl, acetamidomethyl or tert-butylthio, W is resin, and W is selected from WANG resin, CTC resin or Rink Amide MBHA resin;
the route 1 comprises the following specific steps: dissolving iodine in N, N-dimethylformamide, adding into resin A, reacting for 1-3 hr with nitrogen blowing, cutting polypeptide with shearing liquid, and separating and purifying with high performance liquid chromatography to obtain target product;
the route 2 comprises the following specific steps: cutting off the polypeptide from the resin A by using a shearing liquid, separating the polypeptide without oxidized closed ring by using a high performance liquid chromatography, dissolving the polypeptide in a phosphate buffer solution with the pH value of 7.4-8.0, adding an oxidant for oxidation, reacting for 6-12 hours, and separating and purifying by using the high performance liquid chromatography to obtain a target product, wherein the oxidant is selected from oxidized glutathione, selenocysteine or a mixed solution of the oxidized glutathione and reduced glutathione.
In the invention, the shearing liquid is trifluoroacetic acid in volume ratio: triisopropylsilane: water: 1,2 ethanedithiol i.e. TFA/TIPS/H 2 O/EDT (v: v: v: v =94: triisopropylsilane: water: phenol: 1,2 ethanedithiol, TFA/TIPS/H 2 O/PhOH/EDT (v: v: v = 91.5.
In the invention, C is cysteine, AA is L-type amino acid, and R of resin A 1 And R 2 Is trityl and W is WANG resin.
In the present invention, the synthesis method adopts scheme 1, resin A100 mg is placed in a peptide connecting tube, 1mM iodine dissolved in N, N-dimethylformamide is added, and blowing is performed at room temperatureThe nitrogen was reacted for two hours, washed alternately with N, N-dimethylformamide and dichloromethane, and then the resin was transferred to a small 1.5ml centrifuge tube and 1ml of TFA/TIPS/H was added in a volume ratio of 1ml 2 The reaction was carried out for 1 to 2 hours with shaking the sheared solution of O/EDT (v: v: v: v = 94.
In the present invention, the synthesis method adopts scheme 2, 100mg of resin A is placed in a 1.5ml centrifuge tube, and 1ml of TFA/TIPS/H with volume ratio is added 2 The reaction is carried out for 1 to 2 hours by reversed shaking of shearing liquid of O/EDT (v: v: v: v = 94.
According to the invention, the resin A adopts a solid-phase synthesis method based on Fmoc protecting groups, the W resin is selected from WANG resin, CTC resin and Rink Amide MBHA resin, the condensing agent is selected from 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate, 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate, O-benzotriazol-tetramethylurea hexafluorophosphate, benzotriazol-1-yl-oxypyrrolidinophosphonium hexafluorophosphate and 1-hydroxybenzotriazole dual system, O-benzotriazol-N, N, N ', N' -tetramethylurea tetrafluoroborate and 1-hydroxybenzotriazole dual system, and the acid-binding agent is selected from N, N-diisopropylethylamine, triethylamine and 4-dimethylaminopyridine.
In the invention, the synthesis steps of the resin A are as follows: weighing WANG resin 500mg in a peptide connecting tube, dissolving 570mg of 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate, 261 mu l N, N-diisopropylethylamine, 254mg of amino acid Fmoc-Val-OH in an appropriate amount of N, N-dimethylformamide, then adding the peptide connecting tube, carrying out nitrogen blowing reaction for 1 hour, alternately washing N, N-dimethylformamide and dichloromethane, then adding a 50% morpholine N, N-dimethylformamide solution, carrying out nitrogen blowing reaction for half an hour, alternately washing N, N-dimethylformamide and dichloromethane, adding 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate, N, N-diisopropylethylamine and an amino acid Fmoc-Lys (OtBu) -OH N, N-dimethylformamide solution according to an equivalent weight, carrying out nitrogen blowing reaction for 1 hour, alternately washing N, N-dimethylformamide and dichloromethane, then adding 50% acetic anhydride N, N-dimethylformamide solution, blowing an N-dimethylformamide solution, reacting N-dimethylformamide, N-dimethylformamide solution, N-dimethylformamide, and dichloromethane in turn, and adding N-dimethylformamide to synthesize polypeptide by adding the nitrogen end sequence: n, N-diisopropylethylamine: and the volume ratio of the N, N-dimethylformamide is 0.85.
In the present invention, the target product synthesized according to the penicillamine-based polypeptide disulfide bond synthesis method and the protein are added to a phosphate buffer solution having a pH of 7.4 and incubated in a water bath at 37 ℃ for 6 to 12 hours.
The polypeptide disulfide bond synthesis method based on penicillamine is applied to the fields of drug delivery, chemical probes, targeted covalent inhibitors, antibody-coupled drugs, targeted proteasome degradation systems, protein-protein interaction ligand screening and the like.
The invention provides a method for synthesizing polypeptide containing the disulfide bond structure.
The stability of the disulfide bond under in vitro conditions is verified by high performance liquid chromatography, a liquid chromatography-mass spectrometer and the like.
The invention demonstrates the property of this disulfide bond to target the ring opening of adjacent cysteines to reconstitute a new disulfide bond.
The present invention demonstrates the dependence of the efficiency of targeting the disulfide bond to adjacent cysteine ring openings on the disulfide bond equivalents.
The present invention demonstrates the time dependence of this disulfide bond targeting to open the loop adjacent cysteine.
The present invention demonstrates the stability of the newly formed disulfide bond under reducing conditions after targeted induction of binding.
The invention proves that the disulfide bond does not generate negative influence on the binding capacity of the polypeptide through a fluorescence polarization experiment.
The invention verifies the selectivity of the disulfide bond based on the target-induced open loop reconstruction of a new disulfide bond.
The invention provides a polypeptide disulfide bond synthesis method based on penicillamine, which provides design and synthesis of a stable disulfide bond and uses the stable disulfide bond as an effective covalent reaction warhead in the field of chemical biology. According to the invention, the disulfide bond is successfully developed into a stable Covalent Warhead (Warhead) according to a Target Induced Covalent (TIC) concept while being applied to stabilizing the polypeptide according to the relative stability of the disulfide bond, the stability of the disulfide bond in the presence of a corresponding reducing agent is verified, the specificity of the disulfide bond adjacent to a driving open loop (Proximant drive open loop) and a Target point is verified, and example verification and more ideas are provided for the following more chemical and biological applications of the disulfide bond, such as the application in the fields of developing a specific stable Covalent inhibitor (covalence inhibitor), an antibody coupling drug (ADC), a chemical probe and the like. The disulfide bond is introduced into the modification of the stable polypeptide, and simultaneously, the disulfide bond is skillfully applied to the chemical biological research of reconstructing a new disulfide bond near cysteine, so that the biological function of the disulfide bond is further expanded, and a thought is provided for more subsequent biological applications.
Drawings
FIG. 1 is a time-variant LCMS analysis of the polypeptide Ac-Cyclo (PenSPC) NIYYKV-COOH (MW: 1257) in the presence of GSH.
FIG. 2 is a graph of the polypeptide Ac-Cyclo (PenSPC) NIYYKV-COOH (MW: 1257) analyzed by LCMS at different times in the presence of Pys, where 1 is 6 hours; 2 is 24 hours; 3 was 48 hours.
FIG. 3 is a 12-hour LCMS analysis of the polypeptide Ac-QSPANCYCLO (PenYPen) KV-COOH (MW: 1208) in the presence of GSH.
FIG. 4 is a LCMS analysis of the polypeptide Ac-cycle (CSPAPen) IYYKV-COOH (MW: 1215) reacted in the presence of alkylating agent for 24 hours.
FIG. 5 shows LCMS analysis of the polypeptide Ac-cycle (CSPPen) MIYYKV-COOH (MW: 1275) reacted in the presence of alkylating agent for 24 hours.
FIG. 6 shows the chemical formula of the polypeptide Ac-cycle (CSPPen) MIYYKV-COOH (MW: 1275) after alkylation.
FIG. 7 is a SDS-PAGE pattern of different polypeptides and model protein PDZ after 12 hours incubation in PBS. Wherein the polypeptides are respectively: 1,Ac-PenSPANIYKV-COOH (Redox); 2,Ac-CSPANIYYKV-CCOH (Redox); 3,Ac-PenSPACIYKV-COOH (Redox); 4,Ac-CSPAPenIYKV-COOH (Redox); 5,Ac-PenSPCNIYKV-COOH (Redox); 6,Ac-CSPACIYYKV-COOH (Redox); 7,Ac-QSPACIYYKV-COOH (Redox + Cystein); 8,Ac-QSPACIYYKV-COOH (Redox + Pen); 9,Ac-QSPAPenIYKV-COOH (Redox + Cys); 10,Ac-QSPANPENYCKV-COOH (Redox); 11,Ac-QSPANPENYKV-COOH (Redox); 12,Ac-QSPANIYPenKV-COOH (Redox).
FIG. 8 is a SDS-PAGE pattern of the polypeptide Ac-Cyclo (CSPPen) NIYYKV-COOH incubated with PDZ (5. Mu.M) for 4 hours at different concentrations.
FIG. 9 shows SDS-PAGE patterns of the polypeptide Ac-cycle (CSPPen) NIYYKV-COOH (50. Mu.M) incubated with PDZ (5. Mu.M) protein for different periods of time.
FIG. 10 is a graph showing that the polypeptide Ac-cycle (CSPPen) NIYYKV-COOH was reduced in the presence of GSH at various concentrations after binding to PDZ.
FIG. 11 shows FP and Kd values of model proteins PDZ (wild type) for PDZ-4, PDZ-4R, PDZ-6 and PDZ-6R polypeptides, respectively.
FIG. 12 shows FP plots and Kd values for PDZ-4, PDZ-4R, PDZ-6R polypeptides and model protein PDZ (C73S), respectively.
FIG. 13 shows FP plots and Kd values for PDZ-4, PDZ-4R, PDZ-6R polypeptides and model protein PDZ (C33 SC 34S), respectively.
FIG. 14 shows the general structure of disulfide bonds in polypeptides.
Figure 15 is a SDS-PAGE gel diagram of polypeptides containing penicillamine-based disulfide bonds that specifically target PDZ proteins selectively covalently bind to PDZ.
Detailed Description
The present invention is further described with reference to the following examples and figures, which are not intended to limit the scope of the invention, and all such similar substitutes and modifications apparent to those skilled in the art are deemed to be included in the invention. The experimental materials referred to in the following examples are commercially available or can be obtained by a preparation method conventional in the art without specific description. The English abbreviations used in the application documents have the corresponding Chinese meanings as shown in Table 1.
TABLE 1 English abbreviations
Example 1 Penicilliamine-based Synthesis of polypeptide disulfide bonds
1. Synthesis of resin A with WANG resin
Solid phase synthesis of polypeptide (taking WANG resin as an example): WANG resin was weighed into a pipette, DCM was added, and nitrogen was bubbled for 10 minutes to swell. The Fmoc protecting group was removed by addition of 50% (v/v) morpholine in DMF and nitrogen sparge for 30 minutes (twice). The resin was washed with DMF and DCM alternately 8 times and the corresponding Fmoc-AA-OH (5 eq), HCTU (5 eq), DIPEA (10 eq) dissolved in DMF was added to the resin and the reaction was allowed to proceed under nitrogen bubbling for 1 hour. Washing with DMF and DCM for 8 times, and removing protection to obtain another amino acid and polypeptide sequence.
2. Synthesis and isolation of disulfide-bond containing polypeptides (two main routes are exemplified):
route 2: the resin synthesized in example 1 (100 mg) was put in a 1.5ml centrifuge tube, and 1ml of TFA/TIPS/H was added 2 O/EDT (v: v: v: v =94 2 O/PhOH/EDT (v: v: v = 91.5. Utilizing jellyThe polypeptide product is lyophilized in a dry machine to obtain powder, then dissolved in PBS with pH value of 7.4 (a small amount of DMSO can be added), added with 1mM GSSG, reacted overnight at room temperature, and the solution is filtered by a filter membrane, and then separated by high performance liquid chromatography to obtain the target product.
Route 1: the resin (100 mg) obtained in example 1 above was placed in a peptide-connecting tube, 1mM iodine in DMF was added, nitrogen gas was blown at room temperature for two hours, DMF and DCM were alternately washed 8 times, the resulting dried polypeptide was placed in a 1.5ml centrifuge tube, 1ml of the above-mentioned shearing solution in 1 was added and the reaction was reversed and shaken for 1-2 hours, the resin was filtered off, nitrogen gas was blown to volatilize the shearing solution, 0.5ml of precooled ether was then added for precipitation, the supernatant was centrifuged and discarded, the precipitated polypeptide was left at room temperature for 1-9 minutes to sufficiently volatilize ether, and the objective polypeptide was then isolated by high performance liquid chromatography (this time, the product was the objective polypeptide with disulfide bond formed).
The method for synthesizing the disulfide bond-containing polypeptide Ac-Cyclo (PenSPC) NIYYKV-COOH (MW: 1257) according to the steps of 1 and 2 is as follows: weighing WANG resin 500mg (calculated as resin loading degree 0.3 mmol/g) in a peptide connecting tube, dissolving 570mg HATU,261 μ l DIPEA,254mg amino acid Fmoc-Val-OH in appropriate amount of DMF, adding the peptide connecting tube, performing nitrogen blowing reaction for 1 hour, alternately washing DMF and DCM for 8 times, adding 50% morpholine DMF solution, performing nitrogen blowing reaction for half an hour (twice), alternately washing DMF and DCM for 8 times, equivalently adding DMF solution of HATU, DIPEA and amino acid Fmoc-Lys (OtBu) -OH, performing nitrogen blowing reaction for 1 hour, alternately washing DMF and DCM for 8 times, subsequently adding 50% morpholine DMF solution, performing nitrogen blowing reaction for half an hour (twice), and alternately washing DMF and DCM for 8 times. And by analogy, sequentially adding amino acids from a carbon end to a nitrogen end until the synthesis of the polypeptide sequence is completed, and adding acetic anhydride: n, N-diisopropylethylamine: and (3) carrying out a nitrogen blowing reaction for half an hour on a mixed solution of DMF (0.85 to 16) and DMF (16) to block the amino group at the nitrogen terminal of the polypeptide with acetyl, and obtaining the polypeptide according to the method described in the above scheme 2.
The synthesis of the disulfide bond-containing polypeptide Ac-QSPANCYCLO (PenYPen) KV-COOH (MW: 1208) according to the procedures of 1 and 2 was: weighing WANG resin 500mg (calculated as resin loading degree 0.3 mmol/g) in a peptide connecting tube, dissolving 570mg HATU,261 μ l DIPEA,254mg amino acid Fmoc-Val-OH in appropriate amount of DMF, then adding the peptide connecting tube, performing nitrogen blowing reaction for 1 hour, alternately washing DMF and DCM, then adding 50% morpholine DMF solution, performing nitrogen blowing reaction for half an hour (reaction twice), alternately washing DMF and DCM, adding HATU, DIPEA and amino acid Fmoc-Lys (OtBu) -OH in terms of equivalent amount of DMF solution for nitrogen blowing reaction for 1 hour, alternately washing DMF and DCM for 8 times, then adding 50% morpholine DMF solution, performing nitrogen blowing reaction for half an hour (reaction twice), and alternately washing DMF and DCM for 8 times. And adding amino acids from the carbon end to the nitrogen end in sequence by analogy until the synthesis of the polypeptide sequence is completed, and adding acetic anhydride: n, N-diisopropylethylamine: and (3) carrying out a nitrogen blowing reaction for half an hour on a mixed solution of DMF (0.85 to 16) and DMF (16) to block the amino group at the nitrogen terminal of the polypeptide with acetyl, and obtaining the polypeptide according to the method described in the above scheme 1.
The disulfide bond-containing polypeptide Ac-cycle (CSPAPen) IYYKV-COOH (MW: 1215) was synthesized according to the procedures of 1 and 2. The method comprises the following steps: weighing 500mg WANG resin (calculated by the resin loading degree of 0.3 mmol/g) in a peptide connecting tube, dissolving 570mg HATU,261 mu l DIPEA and 254mg amino acid Fmoc-Val-OH in an appropriate amount of DMF, then adding the peptide connecting tube, carrying out nitrogen blowing reaction for 1 hour, alternately washing DMF and DCM, then adding 50% morpholine DMF solution, carrying out nitrogen blowing reaction for half an hour (twice), alternately washing DMF and DCM, equivalently adding the DMF solution of HATU, DIPEA and Fmoc-Lys (OtBu) -OH, carrying out nitrogen blowing reaction for 1 hour, alternately washing DMF and DCM for 8 times, then adding 50% morpholine DMF solution, carrying out nitrogen blowing reaction for half an hour (twice), and alternately washing DMF and DCM for 8 times. And by analogy, sequentially adding amino acids from a carbon end to a nitrogen end until the synthesis of the polypeptide sequence is completed, and adding acetic anhydride: n, N-diisopropylethylamine: 3.15, and performing nitrogen blowing reaction for half an hour to seal the nitrogen terminal amino group of the polypeptide with acetyl, and obtaining the polypeptide according to the method described in the above scheme 2
The synthesis of the disulfide bond-containing polypeptide Ac-cycle (CSPPen) MIYYKV-COOH (MW: 1275) according to the procedures of 1 and 2 was: weighing 500mg WANG resin (calculated by the resin loading degree of 0.3 mmol/g) in a peptide connecting tube, dissolving 570mg HATU,261 mu l DIPEA and 254mg amino acid Fmoc-Val-OH in an appropriate amount of DMF, then adding the peptide connecting tube, carrying out nitrogen blowing reaction for 1 hour, alternately washing DMF and DCM, then adding 50% morpholine DMF solution, carrying out nitrogen blowing reaction for half an hour (twice), alternately washing DMF and DCM, equivalently adding the DMF solution of HATU, DIPEA and Fmoc-Lys (OtBu) -OH, carrying out nitrogen blowing reaction for 1 hour, alternately washing DMF and DCM for 8 times, then adding 50% morpholine DMF solution, carrying out nitrogen blowing reaction for half an hour (twice), and alternately washing DMF and DCM for 8 times. And by analogy, sequentially adding amino acids from a carbon end to a nitrogen end until the synthesis of the polypeptide sequence is completed, and adding acetic anhydride: n, N-diisopropylethylamine: 3.15, and performing nitrogen blowing reaction for half an hour to seal the nitrogen terminal amino group of the polypeptide with acetyl, and obtaining the polypeptide according to the method described in the above scheme 2
The design method and the route of the invention are provided as a disulfide bond structure formed between penicillamine and cysteine, and the route is as follows:
The route when disulfide-bond containing polypeptides react with proteins is shown below:
when the polypeptide of the target protein targets the protein to enable the disulfide bond formed by the penicillamine on the polypeptide to be close to the cysteine near the binding site of the target protein, the disulfide bond is reconstructed, a new disulfide bond is formed with the cysteine, and then the sequence is combined with the protein in a firmer covalent mode.
Example 2 stability validation of disulfide bond containing polypeptides in the presence of a reducing agent:
the polypeptide (1 mM) synthesized according to the method of example 1 was dissolved in 100mM PBS (phosphate buffered saline) at pH 7.4, followed by addition of 10mM GSH or Pys, co-incubation was performed in a water bath at 37 ℃, and the reaction solutions after 24 hours and 48 hours of co-incubation were taken, respectively, and analyzed by a liquid chromatography-mass spectrometer to observe reduction of disulfide bonds.
The LCMS analysis chart of the polypeptide Ac-Cyclo (PenSPC) NIYYKV-COOH (MW: 1257) in the presence of GSH at different times is shown in figure 1, namely the analysis and identification chart of the polypeptide by LCMS after incubation with GSH for 24 hours and 48 hours respectively. As can be seen, the polypeptide was not substantially reduced.
The profile of the LCMS analysis of the polypeptide Ac-Cyclo (PenSPC) NIYYKV-COOH (MW: 1257) in the presence of Pys is shown in FIG. 2, i.e., the profile identified by the LCMS analysis after 6 hours, 24 hours and 48 hours of co-incubation of the polypeptide with 2-mercaptopyridine. As can be seen, the polypeptide was not substantially reduced.
A12 hour LCMS analysis of the polypeptide Ac-QSPANCYCLO (PenYPen) KV-COOH (MW: 1208) in the presence of GSH is shown in FIG. 3, which is a plot of LCMS identification after 12 hours of co-incubation of the polypeptide and GSH. As can be seen, the polypeptide was not substantially reduced.
Example 3 stability verification of disulfide bond containing polypeptides in the presence of alkylating agents (exemplified by 1,3-2 bromomethylbenzene):
the polypeptide synthesized according to the method of example 1 was dissolved in 100mM PBS at pH 7.4, added with a DMSO (2-methyl sulfoxide) dissolved alkylating reagent (1.5 mM), reacted in the presence of 1% formic acid for 24 hours, and then analyzed by a liquid chromatography-mass spectrometer to observe the change of the reaction system.
LCMS analysis of the polypeptide Ac-cycle (CSPAPen) IYYKV-COOH (MW: 1215) reacted for 24 hours in the presence of alkylating agent is shown in FIG. 4, i.e., the polypeptide reacted with alkylating agent 1,3-dibromomethylbenzene for 24 hours in the presence of 1% formic acid is analyzed by LCMS. As can be seen from the figure, the polypeptide is not substantially changed.
The LCMS analysis of the polypeptide Ac-cycle (CSPPen) MIYYKV-COOH (MW: 1275) reacted for 24 hours in the presence of alkylating agent is shown in FIG. 5, i.e., the polypeptide reacted with alkylating agent 1,3-dibromomethylbenzene for 24 hours in the presence of 1% formic acid followed by LCMS analysis. As can be seen from a comparison of FIGS. 4 and 5, the alkylating agent reacts with methionine in the polypeptide.
FIG. 6 shows the chemical formula of the polypeptide Ac-cycle (CSPPen) MIYYKV-COOH (MW: 1275) after alkylation.
Example 4 characterization of disulfide bond-containing polypeptides targeting adjacent cysteine ring opening reconstruction of new disulfide bonds:
the polypeptide synthesized according to example 1 (10. Mu.M) was incubated with the model protein PDZ (1. Mu.M) in 100mM PBS at pH 7.4 in a water bath at 37 ℃ for 12 hours, and subsequently analyzed by SDS-PAGE gel electrophoresis for the formation of new disulfide bonds, i.e.for the formation of new covalent bands. The model protein PDZ protein is a PDZ structure domain of RGS3 protein, the molecular weight is about 11kDa, naked cysteine is at 33, 34 and 73 positions, and adjacent positions are targeted to cysteine at 33 and 34 positions, so the protein is a very good model protein for disulfide bond polypeptide targeting induction of open loop reconstruction covalent bond.
The SDS-PAGE patterns of the different polypeptides and model protein PDZ after 12 hours incubation in PBS are shown in FIG. 7. Wherein the polypeptides are respectively:
1、Ac-PenSPANIYYKV-COOH(Redox)；
2、Ac-CSPANIYYKV-CCOH(Redox)；
3、Ac-PenSPACIYYKV-COOH(Redox)；
4、Ac-CSPAPenIYYKV-COOH(Redox)；
5、Ac-PenSPCNIYYKV-COOH(Redox)；
6、Ac-CSPACIYYKV-COOH(Redox)；
7、Ac-QSPACIYYKV-COOH(Redox+Cysteine)；
8、Ac-QSPACIYYKV-COOH(Redox+Pen)；
9、Ac-QSPAPenIYYKV-COOH(Redox+Cys)；
10、Ac-QSPANPenYCKV-COOH(Redox)；
11、Ac-QSPANPenYYKV-COOH(Redox)；
12、Ac-QSPANIYPenKV-COOH(Redox)。
( Wherein Redox represents a disulfide bond formed within the polypeptide molecule; redox + indicates that the polypeptide forms a disulfide bond with the amino acid in an intermolecular fashion. )
Example 5 specific (selective) validation of disulfide bond containing polypeptides targeting induced ring opening:
the disulfide-bond-containing polypeptide Ac-cycle (CSPPen) MIYYKV-COOH (10. Mu.M) synthesized according to the method of example 1 was incubated with GST and SND1 (1. Mu.M), respectively, in 100mM PBS at pH 7.4 in a water bath at 37 ℃ for 12 hours, and then analyzed by SDS-PAGE gel electrophoresis whether a new covalent band was formed. The polypeptide is a targeting polypeptide of a model protein PDZ and has no binding capacity with other proteins.
As shown in FIG. 15, it is a SDS-PAGE picture of the polypeptide Ac-cycle (CSPPen) MIYYKV-COOH (10 μ M) incubated with different proteins
Example 6 validation of disulfide bond containing polypeptide targeting adjacent cysteine ring opening efficiency and polypeptide equivalent relationship:
the polypeptides synthesized according to the method of example 1 were incubated with model protein PDZ (1. Mu.M) in 100mM PBS at pH 7.4 in water bath at 37 ℃ for 12 hours in 0.5, 1,2, 3, 5, 10 equivalents, respectively, and subsequently analyzed by SDS-PAGE gel electrophoresis for the formation of new disulfide bonds, i.e., new covalent bands.
FIG. 8 is a SDS-PAGE pattern of the polypeptide Ac-Cyclo (CSPPen) NIYYKV-COOH incubated with PDZ (5. Mu.M) for 4 hours at different concentrations.
Example 7 disulfide bond containing polypeptide targeting adjacent cysteine ring opening time dependent validation:
the polypeptides synthesized according to example 1 (10. Mu.M) and the model protein PDZ (1. Mu.M) were incubated in 100mM PBS at pH 7.4 in a water bath at 37 ℃ for 0, 10, 30, 60, 240, 360, 720 minutes, respectively, and subsequently analyzed by SDS-PAGE gel electrophoresis for the formation of new disulfide bonds, i.e.for the formation of new covalent bands.
FIG. 9 is a SDS-PAGE pattern of the polypeptide Ac-cycle (CSPPen) NIYYKV-COOH (50. Mu.M) incubated with PDZ (5. Mu.M) protein for various periods of time.
Example 8 stability of newly formed disulfide bonds targeting adjacent cysteines under reducing agent conditions:
the polypeptide synthesized according to the method of example 1 (10. Mu.M) and the model protein PDZ (1. Mu.M) were incubated in 100mM PBS at pH 7.4 in a water bath at 37 ℃ for 12 hours, followed by addition of GSH at concentrations of 0, 0.05, 0.1, 0.5, 1, 5, 10mM, respectively, for further incubation for 12 hours, and then analyzed for newly disulfide bond reduction by SDS-PAGE gel electrophoresis.
FIG. 10 is a graph showing that the polypeptide Ac-cycle (CSPPen) NIYYKV-COOH was reduced in the presence of GSH at various concentrations after binding to PDZ.
Example 9 verification of the binding ability of disulfide bond-containing polypeptides to a target protein:
the verification adopts a Fluorescence Polarization (FP) experiment, namely, the binding capacity of the linear polypeptide with the fluorescent group and the disulfide bond-containing polypeptide with the model protein PDZ is respectively determined. Specifically, the target protein was diluted in a gradient manner to obtain a protein series of concentrations of 0 to 100. Mu.M, the fluorescent-group-containing polypeptide synthesized according to the method of example 1 was added to a final concentration of 10nM, followed by incubation at room temperature for ten minutes, the corresponding value was read by the FP program in a microplate reader, the obtained value was introduced into GraphPad software, and a one-site competitive model was selected for nonlinear fitting to obtain a Kd value. The buffer used in the above experiment was 100mM PBS at pH 7.4 and the containers were universal 384 well plates.
FIG. 11 shows FP and Kd values of model proteins PDZ (wild type) for PDZ-4-, PDZ-4R, PDZ-6, and PDZ-6R polypeptides, respectively.
FIG. 12 shows FP plots and Kd values for PDZ-4, PDZ-4R, PDZ-6R polypeptides and model protein PDZ (C73S), respectively.
FIG. 13 shows FP plots and Kd values for PDZ-4, PDZ-4R, PDZ-6R polypeptides and model protein PDZ (C33 SC 34S), respectively.
The polypeptides are respectively:
PDZ-4：FITC-AHX-CSPPenNIYYKV-COOH；
PDZ-4R：FITC-AHX-Cyclo(CSPPen)NIYYKV-COOH；
PDZ-6：FITC-AHX-CSPAPenIYYKV-COOH；
PDZ-6R：FITC-AHX-Cyclo(CSPAPen)IYYKV-COOH。
fluorescence polarization experiments prove that the disulfide bond does not negatively influence the binding capacity of the polypeptide.
Example 10 this disulfide bond was used to modify FK228 application examples:
based on the above concept, the specific operation is to replace cysteine in FK228 by penicillamine according to the existing synthetic route of synthetic natural product FK228, and then obtain the compound with the structure shown in 2, so as to improve or improve the related physicochemical properties of FK 228. Synthetic FK228 pathways have been reported in the literature and patents and are not described in detail herein.
Claims (5)
1. A polypeptide disulfide bond synthesis method based on penicillamine, the structural general formula of the polypeptide disulfide bond is shown as formula (I),
c is cysteine or penicillamine, n is the number of AA, n is 2 or 3, AA represents amino acid without sulfydryl except penicillamine and cysteine, pen is penicillamine, characterized in that the synthesis method adopts the scheme 1 or the scheme 2,
wherein R of the resin A 1 And R 2 Is a mercapto protecting group selected from one of trityl, acetamidomethyl or tert-butylthio, W is resin, and W is selected from WANG resin, CTC resin or Rink Amide MBHA resin;
the route 1 comprises the following specific steps: resin A100 mg was placed in a linker tube, 1mM iodine in N, N-dimethylformamide was added, nitrogen was bubbled at room temperature for two hours, the reaction was washed alternately with N, N-dimethylformamide and dichloromethane, then the resin was transferred to a 1.5ml centrifuge tube and 1ml of trifluoroacetic acid: triisopropylsilane: water: 1,2-ethanedithiol is a 94, 2.5, shear fluid is stirred upside down for 1-2 hours, the resin is filtered and removed, nitrogen is blown to volatilize the shear fluid, then 0.5ml of precooled ether is added for precipitation, the supernatant is removed by centrifugation, the mixture is placed at room temperature for 8 minutes to fully volatilize the ether, and then the target product is separated by high performance liquid chromatography;
the route 2 comprises the following specific steps: 100mg of resin A was placed in a 1.5ml centrifuge tube and 1ml of trifluoroacetic acid: triisopropylsilane: water: 1,2-ethanedithiol is 94.
2. The method for synthesizing a polypeptide disulfide bond according to claim 1, wherein: c is cysteine, AA is L-amino acid, R of resin A 1 And R 2 Is trityl and W is WANG resin.
3. The method for synthesizing a polypeptide disulfide bond according to claim 1, wherein: the resin A adopts a solid-phase synthesis method based on Fmoc protecting groups, W is selected from WANG resin, CTC resin or Rink Amide MBHA resin, the condensing agent is selected from 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate, 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate, O-benzotriazol-tetramethylurea hexafluorophosphate, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate and 1-hydroxybenzotriazole dual system, O-benzotriazol-N, N, N ', N' -tetramethylurea tetrafluoroborate and 1-hydroxybenzotriazole dual system, and the acid-binding agent is selected from N, N-diisopropylethylamine, triethylamine and 4-dimethylaminopyridine.
4. The method for synthesizing polypeptide disulfide bond according to claim 1, wherein the step of synthesizing resin A is: weighing WANG resin 500mg in a peptide connecting tube, dissolving 570mg of 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate, 261 mu l N, N-diisopropylethylamine, 254mg of amino acid Fmoc-Val-OH in an appropriate amount of N, N-dimethylformamide, then adding the peptide connecting tube, performing nitrogen blowing reaction for 1 hour, alternately washing N, N-dimethylformamide and dichloromethane, then adding 50% of morpholine N, N-dimethylformamide solution, performing nitrogen blowing reaction for half an hour, alternately washing N, N-dimethylformamide and dichloromethane, adding 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea hexafluorophosphate, N, N-diisopropylethylamine and amino acid Fmoc-Lys (OtBu) -OH in equivalent amount, performing nitrogen blowing reaction for 1 hour, alternately washing N, N-dimethylformamide and dichloromethane, then adding 50% of N, N-dimethylformamide, blowing N-dimethylformamide solution, performing nitrogen blowing reaction for half an hour, sequentially adding N, N-dimethylformamide solution and dichloromethane to synthesize polypeptide by adding the sequence: n, N-diisopropylethylamine: n, N-dimethylformamide volume ratio of 0.85.
5. The use of the method for synthesizing polypeptide disulfide bonds based on penicillamine according to claim 1, wherein the target product synthesized according to the method of claim 1 and the target protein are added into phosphate buffer with a pH value of 7.4, and incubated in water bath at 37 ℃ for 6-12 hours, so that the target product and the target protein form new disulfide bonds, wherein C is cysteine, and naked cysteine exists near the binding site of the target protein.
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| CN106366160A (en) * | 2016-10-11 | 2017-02-01 | 厦门大学 | Method for establishing polypeptide molecular skeleton rich in disulfide bond based on disulfide bond precise pairing |
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| Application of acetamidomethyl and 9-fluorenylmethyl groups for efficient side protection of penicillamine in solid-phase peptide synthesis;CARLOS GARCIA-ECHEVERRiA et al.;《INTERNATIONAL JOURNAL OF PEPTIDE AND PROTEIN RESEARCH》;19900531;第35卷(第5期);摘要、第409页右栏第2-3段、第411段左栏第3 * |
| Cyclization of disulfide-containing peptides in solid-phase synthesis;FERNANDO ALBERICIO et al.;《INTERNATIONAL JOURNAL OF PEPTIDE AND PROTEIN RESEARCH》;19910501;第37卷(第5期);第439页左栏第1-3段 * |
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