CN102827251B - Transmembrane peptide-mediated antisense antibacterial agent and preparation method and application thereof - Google Patents
Transmembrane peptide-mediated antisense antibacterial agent and preparation method and application thereof Download PDFInfo
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- CN102827251B CN102827251B CN201210335017.3A CN201210335017A CN102827251B CN 102827251 B CN102827251 B CN 102827251B CN 201210335017 A CN201210335017 A CN 201210335017A CN 102827251 B CN102827251 B CN 102827251B
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Abstract
The invention discloses a transmembrane peptide-mediated antisense antibacterial agent which comprises popliteal lymph node assay (PLNA) 787; and the PLNA 787 is formed by connecting transmembrane peptide (KFF) 3K and LNA 787 by cysteine-(4-(N-maleimide methyl)-1- cyclohexidine actidione)-hexane and is prepared by a solid phase synthesis method. The transmembrane peptide-mediated antisense antibacterial agent takes bacteria curing gene as a target spot, has the advantages of being high in specificity, efficient, low in toxicity, safe and the like, has remarkable inhibition effect on the growth of drug-resistant bacteria, and especially has remarkable antagonism on the growth of methicillin-resistant staphylococcus aureus.
Description
Technical field
The invention belongs to technical field of molecular biology, particularly peptide-mediated antisense antibacterial agent of a kind of permeable membrane and its preparation method and application.
Background technology
Streptococcus aureus is that nosocomial infection and community obtain the most common, the most important pathogenic bacterium of infection, and along with antibiotic widespread use, the resistance situation of golden Portugal bacterium is more and more serious.At present, more than 90% golden Portugal bacterium is to Penicillin-resistant, from Britain's reported first methicillin-resistant staphylococcus aureus (methicillin resistant staphylococcus aureus in 1961, MRSA) so far, extend over the entire globe of the infection being caused by MRSA, and its recall rate constantly increases.MRSA is to all β-lactam antibiticss and other multiple antibiotic resistances, and the infection therefore being caused by MRSA is treated very difficultly, and mortality ratio is very high.The most effective medicine for the treatment of MRSA is vancomycin at present, but along with the appearance of vancomycin resistance MRSA, people are about to face the condition that can use without medicine, and therefore finding novel targets that the anti-MRSA of new specificity infects and New Policy becomes study hotspot and the difficult point in bacterium treatment of infection field.
The research and development of traditional antibacterials are mainly that known microbiotic is carried out to structural modification, but find that according to the method new primer becomes more and more difficult, bacterium produces the speed of resistance considerably beyond the research and development speed of new antimicrobial agent simultaneously, so must find new breakthrough mouth.Produce the speed of resistance owing to having the antibacterials of the brand-new binding mode bacterium that can greatly slow down, therefore the research of antibacterials at present mainly concentrates on the medicine of finding new action target and new binding mode.The antisense antibacterial agent that AVI company of the U.S. in 2005 proposes is a kind of antibacterials with brand-new binding mode---the degraded of the target RNA that mediates by RNase H and suppress the copying and transcribe and transcribe of DNA after the mode such as processing and translation suppress growth, the breeding of the transcribing of pathogenic bacterium gene, translation and then anti-bacteria, thereby reach the object of attenuation, inhibition or kill bacteria, such medicine is the novel antibacterial medicine of tool research and development potential.
Compared with conventional medicament, cure the disease gene as the antisense antibacterial agent of target spot is because having the features such as high specific, high efficiency, low toxicity, safety take bacterium, be the novel antibacterial medicine that has exploitation potential.In the antisense molecule of numerous kinds, lock nucleic acid (LNA) has caused people extensive concern as a kind of nucleotide derivative of novelty, promise to be the breach of molecular level treatment various diseases.LNA is a kind of special double-ring oligonucleotide derivative, 2 of structure amplifying nucleic acid '-O, 4 '-C position forms Oxymethylene bridge, sulphur methylene bridge or amine methylene bridge by different shrink effects, and connect into annular, form the condensation structure of rigidity, increased the stability of phosphate backbone local structure.Because LNA structurally has identical phosphate backbone with DNA/RNA, so it has good recognition capability and powerful avidity to DNA, RNA, compared with other oligonucleotide analogs, LNA has many good qualities: 1) and the two strands of DNA, RNA complementation have very strong thermostability; 2) there is the stability that resists 3 ' deoxynucleotide enzyme liberating; 3) LNA-DNA hybrid can activate RNase H; 4) free of toxic effects in body; 5) have oligomerization effect automatically efficiently, synthetic method is relatively simple, and the few nucleic acid chains available amino end of the LNA phosphoric acid method of partially or completely modifying is synthetic on automatic dna synthesizer, and as can be seen here, LNA is a kind of desirable antisense drug.
But, the prerequisite that LNA gives full play to its antisense effect is to reach effective concentration by the said target mrna combining site in bacterial cell, and LNA is the macromolecular cpd of bear electric charge, be difficult for seeing through the cell walls of bacterium and cytolemma, intracellular concentration is low, and can nonspecificly be absorbed by body cell, therefore the drug carrier of passing efficient, selectively targeted bacterium is the bottleneck problem that the agent of restriction antisense antibacterial is developed.
Summary of the invention
The object of the invention is to by the fissional key protein of MRSA---thread temperature sensitive albumen Z(flamentous temperature-sensitive protein Z, FtsZ) structure gene ftsZ mRNA is that target spot is studied, and peptide-mediated antisense antibacterial agent of a kind of permeable membrane and its preparation method and application is provided.
The object of the present invention is achieved like this:
The antisense antibacterial agent that permeable membrane is peptide-mediated, comprises (KFF)
3k-Cys-SMCC-C
6-LNA787, described (KFF)
3k-Cys-SMCC-C
6-LNA787 is permeable membrane peptide (KFF)
3between K and LNA787, be connected with halfcystine-(4-(N-maleimide methyl)-1-Cyclohexamide)-hexane;
The sequence of described LNA787 is 5'-T+*GA+CT*C+GCC*A+C+CA*GTAA*TA+T+*T-3', and wherein A is adenosine acid mono, and T is thymidylic acid monomer, and C is cytidine(C acid mono, and G is guanosine-acid mono; * X represents thio-modification, and X+ represents that LNA modifies, and X is A, T, C or G;
The Nucleotide of described thio-modification is as shown in formula I, and in the nucleotide structure that described LNA modifies, 2 '-O position and 4 '-C position forms Oxymethylene bridge by shrink effect, and connects into annular, as shown in formula II:
Described permeable membrane peptide (KFF)
3the sequence of K is 5'-KFFKFFKFFK-3', and wherein K is Methionin, and F is phenylalanine.
Described (KFF)
3k-Cys-SMCC-C
6the sequence of-LNA787 is as follows:
5'-KFFKFFKFFK-Cys-SMCC-C
6-T+*GA+CT*C+GCC*A+C+CA*GTAA*TA+T+*T-3'。
The antisense antibacterial agent that above-mentioned permeable membrane is peptide-mediated, wherein the base in nucleotide monomer is guanine, VITAMIN B4, cytosine(Cyt), thymus pyrimidine.
The present invention also provides the preparation method of the peptide-mediated antisense antibacterial agent of above-mentioned permeable membrane, wherein said (KFF)
3k-Cys-SMCC-C
6permeable membrane peptide (KFF) 3K and LNA787 are passed through the Cys-SMCC-C as spaced interval by-LNA787
6covalently bound, obtain with solid-phase synthesis preparation.
The present invention is by experimental study (KFF)
3k-Cys-SMCC-C
6-LNA787(is hereinafter referred to as PLNA787) impact on Mu50 colony number, result shows that PLNA787 can significantly reduce the colony number of Mu50, this restraining effect has concentration dependent.In addition, the present invention is the impact on Mu50 growth by experimental study PLNA787, and result shows the growth of the inhibition Mu50 of PLNA787 energy concentration dependent.According to these test-results, the invention provides the application of the peptide-mediated antisense antibacterial agent of above-mentioned permeable membrane in the medicine of preparing antimicrobial agent.Preferably described resistant organism is methicillin-resistant staphylococcus aureus.
The peptide-mediated antisense antibacterial agent tool of permeable membrane involved in the present invention has the following advantages and is progressive significantly: take bacterium Disease-causing gene as target spot, have the advantages such as high specific, high efficiency, low toxicity, safety.The growth of resistant organism is had to significant restraining effect, especially significantly the growth of antagonism methicillin-resistant staphylococcus aureus.Be in particular in: the test-results by embodiment 4 can find out, PLNA787 can significantly reduce the colony number of Mu50, also can suppress the growth of Mu50; RT-PCR method detects to be found, naked LNA can not suppress the expression of the ftsZ gene of MRSA bacterial strain, and the PLNA787 of lower concentration can suppress the ftsZ genetic expression of MRSA bacterial strain completely.
Accompanying drawing explanation
The molecular structure of Fig. 1 PLNA.
The colony number of the various PLNA of Fig. 2 on Mu50 affect histogram; * P<0.01vs.control.
The color atlas of Fig. 3 HPLC method purifying LNA787.
The MALDI-TOF mass spectroscopy figure of Fig. 4 LNA787.
The color atlas of Fig. 5 RP-HPLC method purifying PLNA787.
The MALDI-TOF mass spectroscopy figure of Fig. 6 PLNA787
The histogram of the colony number of Fig. 7 PLNA787 to Mu50; * P<0.05vs.control; * P<0.01vs.control.
The influence curve figure of Fig. 8 PLNA787 to Mu50 growth.
The affect electrophorogram of Fig. 9 PLNA787 on ftsZ genetic expression.
Embodiment
The present invention is with the fissional key protein of regulation and control MRSA---thread temperature sensitive albumen Z(filamentous temperature-sensitive protein Z, FtsZ) structure gene ftsZ mRNA is target spot, design, screen and synthesize lock nucleic acid (the locked nucleic acid of selectively targeted ftsZ mRNA, LNA), and by permeable membrane peptide and the covalently bound PLNA of LNA, in body, the effect that Pharmacodynamics in vitro experiment discussion PLNA treatment MRSA infects, and finishing screen has been selected the significant LNA787 of effect, its can be separately as or become with vehicle preparation newtype drug---the antisense antibacterial agent that the anti-MRSA of specificity infects, for new breakthrough mouth has been found out in the control that MRSA infects.
Below the Preparation Example of PLNA and the effect test example of PLNA787 the present invention relates to.
(1) Effective target site of screening ftsZ mRNA, the LNA of design differential high efficient: adopt the sequence of RNA Structure4.5 software detailed analysis ftsZ mRNA and predict its secondary structure, take 18-21 base as LNA elementary length, in non-conservative sequence area, non-folding region selects Effective target site, filters out 10 optimum LNA(in table 1).
Table 1 is for the lock nucleic acid (LNA) of ftsZ mRNA design
Note: in nucleotide sequence hurdle, capitalization represents LNA, lowercase representation DNA.
Preparation and the separation and purification of embodiment 2 PLNA
(1) preparation of permeable membrane peptide (KFF) 3K: synthetic by fluorenylmethyloxycarbonyl solid phase synthesis process (Fmoc-SPPS); take HATU/DIEA as condensing agent; on MBHA-Rink resin; adopt Fmoc protection a-amino acid; the polypeptide that end (aminoterminal) solid phase synthesis C-end is halfcystine from C-end (carboxyl terminal) to N-, synthetic polypeptide is sheared and passes through RP-HPLC separation and purification through the TFA of high density.The aminoacid sequence of permeable membrane peptide (KFF) 3K is KFFKFFKFFK, and K is Methionin (Lys), and F is phenylalanine (Phe).
Concrete steps are: the first step, and the solid phase synthesis of polypeptide: the polypeptide of preparation holds N end to carry out one by one from C, first MBHA-Rink resin dress post, (CHCl adds methylene chloride
2), make it swelling, 20% piperidines/dimethyl formamide (DMF) solution deprotection, DMF cleans.First amino acid, the 2-(7-azo benzotriazole 9-fluorenylmethyloxycarbonyl (Fmoc) protected with the mol ratio of 1:1:2)-N; N; N '; N '-tetramethyl-urea phosphofluoric acid ester (HATU) and N; N-diisopropylethylamine (DIEA) is dissolved in appropriate DMF; resin after activation and after deprotection is at post cocycle coupled reaction 2 ~ 6h, and DMF washs.The amino acid of follow-up connection repeats the process of above-mentioned activation, condensation, deprotection and washing, until preparation finishes.Second step, the shearing of polypeptide: take off reacted resin, add and shear liquid (95% trifluoroacetic acid+5% meta-cresol), room temperature reaction 1.5-2h, filters, and adds the anhydrous diethyl ether of the precooling of 10 times of volumes in filtrate, the centrifugal 5min of 3500rpm, collecting precipitation drying at room temperature.The 3rd step: the separation and purification of polypeptide: weigh a certain amount of dried polypeptide, be dissolved in 0.1% trifluoroacetic acid, through reverse post gradient separations (elutriant is the acetonitrile containing the 20-80% of 0.1% trifluoroacetic acid), collect 260nm place elution peak after sample preparation.
(2) preparation of lock nucleic acid (LNA): adopt the solid phase phosphorous acid acid amides method of standard synthetic.
Concrete steps are: first by oligonucleotide chain 3 ' end nucleosides (N1) with its 3 '-OH by 1 long alkyl arm and the controlled micropore glass pearl of solid phase carrier (CPG) be coupled, the 5 '-OH of N1 protects with dimethoxytrityl (DMTr).Then start spreading oligonucleotide acid chain step by step from N1.1 synthesis cycle comprises 4 steps.The first step: go protection, remove with trichoroacetic acid(TCA) (TCA) DMTr that CPG connects 5 on Nucleotide '-end, expose 5 '-OH, washing is in order to carrying out the next step.Second step: coupled reaction, before condensation, deoxynucleoside phosphoramidite monomer and tetrazolium are mixed and fed into synthetic post, tetrazolium provides a proton to the N atom of diisopropylamino on 3 ' phosphoric acid, protonated Diisopropylamine is a good free group, form this active intermediate of phosphoramidite tetrazolium with tetrazolium, this step tetrazolium is excessive has guaranteed monomer activation fully.When phosphoramidite tetrazolium contacts with CPG connected Nucleotide N1, there is nucleophilic reaction with its 5 '-OH, condensation occurs and take off tetrazolium, acetonitrile washing.The 3rd step: capping, add aceticanhydride and dimethyl aminopyridine, make not participate under the oligonucleotide chain acetyl of reaction, acetylizad oligonucleotide chain is not participated in next step reaction.The 4th step: oxidizing reaction, add iodine, make the phosphorous acid of trivalent change more stable pentavalent phosphoric acid into.After completing, above-mentioned circulation carries out second synthesis cycle.Every experience one is taken turns circulation, extends 1 Nucleotide.The oligonucleotide chain extending is fixed on insoluble solid phase carrier all the time, and excessive unreacted reactant or resolvent are by filtering or washing and remove.Synthetic and the aforesaid method difference of thio-modification Nucleotide have two: one, and vulcanization reaction replaces oxidizing reaction, and the 2nd, capping completes after vulcanization reaction.The synthetic method of the Nucleotide that LNA modifies is the same, and just nucleotide monomer is the nucleotide monomer of modifying through LNA, directly carries out above-mentioned four-step reaction.
Wherein, the sequence of LNA787 is 5'-T+*GA+CT*C+GCC*A+C+CA*GTAA*TA+T+*T-3', and wherein A is adenosine acid mono, and T is thymidylic acid monomer, and C is cytidine(C acid mono, and G is guanosine-acid mono; * X represents thio-modification, and X+ represents that LNA modifies, and X is A, T, C or G; The Nucleotide of thio-modification is as shown in formula I, in the nucleotide structure that LNA modifies, 2 '-O position and 4 '-C position forms Oxymethylene bridge by shrink effect, and connect into annular, this annular bridge has locked the configuration of furanose, reduce the snappiness of ribose structure, increase the stability of phosphate backbone local structure, as shown in formula II.
Adopt HPLC to carry out separation and purification to LNA crude product, all obtain more than 98% sterling of purity, the molecular weight of measuring sterling with MALDI-TOF conforms to theoretical value.Fig. 3 and Fig. 4 are respectively HPLC figure and the MALDI-TOF figure of synthetic LNA787, and through HPLC purifying, the purity of LNA787 is that the molecular weight of 98.58%, MALDI-TOF mensuration is 6700.28, conforms to theoretical value 6699.24.
(3) permeable membrane peptide (KFF)
3connection and the Purification of K and lock nucleic acid LNA: when oligonucleotide chain reaches after predetermined length, excessive SMCC joins on the oligonucleotide being connected on CPG, hatches 1h, fully washs final vacuum dry.Be connected to oligonucleotide on CPG and excessive permeable membrane peptide overnight incubation altogether, fully washing, (KFF)
3the connector (being PLNA) of K and LNA cuts from solid phase carrier, and fresh strong aqua is sloughed protecting group (ammonia solution), vacuum-drying.PLNA crude product is carried out to purifying by HPLC, and identify by MALDI-TOF mass spectrum.PLNA molecule prepared by the present invention is permeable membrane peptide (KFF)
3the connector of K and LNA787, permeable membrane peptide (KFF)
3between K and LNA with halfcystine-(4-(N-maleimide methyl)-1-Cyclohexamide)-hexane (Cys-SMCC-C
6) connect wherein (KFF)
3k is positioned at halfcystine end, and LNA is positioned at hexane cardinal extremity, and concrete structure is shown in Fig. 1.
Adopt the crude product of the connector (PLNA) of RP-HPLC to permeable membrane peptide and LNA to carry out separation and purification, all obtain more than 90% sterling of purity, the molecular weight of measuring sterling with MALDI-TOF conforms to theoretical value.Fig. 5 and Fig. 6 are respectively RP-HPLC figure and the MALDI-TOF figure of PLNA787, and through RP-HPLC purifying, the purity of PLNA787 is that the molecular weight that 91.13%, MALDI-TOF measures is 8616.14, conforms to theoretical value 8614.85.
The minimal inhibitory concentration research of embodiment 3 PLNA to Mu50
In LNA one of carbon tip, it is passed through to the permeable membrane peptide (KFF) of cell walls with promoting LNA respectively according to the method for embodiment 2
3k is connected, and is prepared into PLNA.
Comprehensive improvement microwell plate MIC measuring method and colony counting method are as the method for effective antibacterial antisense target position screening.First contriver measures (in table 2) to 10 anti-ftsZ PLNA sequences (anti-ftsZ PLNA 01,201,274,309,322,571,593,705,787 and 1153) to the MIC of S.aureus, and result shows that 10 PLNA all have the strong and weak anti-microbial activity not waiting.Again the PLNA of each 10 μ M and Mu50 are hatched to 8h altogether, carry out plate clone experiment, counting CFU, result shows that 10 PLNA all can obviously reduce the colony number (Fig. 2) of Mu50.The experimental result of comprehensive improvement microwell plate MIC measuring method and colony counting method, contriver thinks that PLNA787 has the strongest bactericidal potency.
The minimal inhibitory concentration (MIC) of table 2 PLNA to Mu50
The anti-microbial effect research of embodiment 4 PLNA787 to MRSA bacterial strain Mu50
The mensuration of A.PLNA787 minimal inhibitory concentration (MIC): adopt By Dilution MIC.By the bacterium transferred species of fresh incubated overnight, in nutrient broth medium, 35 ℃, 210rpm are cultured to logarithmic growth mid-term, survey A
630=0.5 ~ 0.6, be diluted to 0.5 Maxwell than turbid standard, then 1:300 dilution.The bacterium liquid of getting 50 μ l joins respectively in the test tube of the PLNA787 that contains doubling dilution, hatches 12～16h for 35 ℃.In every pipe, add 10g/L(1%) TTC (TTC) 10 μ l, 35 ℃ have bacterial growth pipe to take on a red color after hatching 3h, not aobvious red lowest concentration of drug is that PLNA787 is to detecting the MIC value of bacterium, PLNA787 is 1.6 μ M to the MIC value of Mu50, and the MIC value ﹥ 25 μ M of naked LNA787, in table 3.
The minimal inhibitory concentration (MIC) of table 3 PLNA787 to Mu50
Control: blank; PLNA787: the covalent conjunct agent of permeable membrane peptide and LNA787;
Free-LNA787: naked LNA787; Free-Pep: naked (KFF)
3k.
The impact of B.PLNA787 on Mu50 colony number: by the bacterium liquid transferred species of fresh incubated overnight in nutrient broth medium, 35 ℃, 210rpm are cultured to logarithmic growth mid-term, to the PLNA787 that adds different concns in nutrient solution, hatch altogether 10h, enumeration of bacterial colonies number (CFU).Fig. 7 result PLNA787 can significantly reduce the colony number of Mu50, and this restraining effect has concentration dependent.
The impact of C.PLNA787 on Mu50 growth: by the bacterium liquid transferred species of fresh incubated overnight in nutrient broth medium, 35 ℃, 210rpm are cultured to logarithmic growth mid-term, to the PLNA787 that adds different concns in nutrient solution, hatch altogether different time, draw growth curve, observe bacterial growth situation.Fig. 8 result shows the growth of the inhibition Mu50 of PLNA787 energy concentration dependent.
D.RT-PCR detects PLNA787 and ftsZ is expressed to the impact changing: by the bacterium liquid transferred species of fresh incubated overnight, in nutrient broth medium, 35 ℃, 210rpm are cultured to logarithmic growth mid-term, to the PLNA787 that adds different concns in nutrient solution, hatches altogether 6h.Cultivation finishes rear collection bacterium, and Trizol method is extracted the total RNA of bacterium, and reverse transcription obtains cDNA as reaction template, RT reaction conditions: 37 ℃, and 60min, the laggard performing PCR of ice bath.PCR reaction conditions: 95 ℃ of denaturation 2min, 95 ℃ of sex change 30sec, 58 ℃ of annealing 30sec, 72 ℃ are extended 30sec, 30 circulations.PCR primer: 5 '-ACCACGGAATGAATAA-3 ' and 5 '-CCTGCTCCTAAACCAC-3 '.Fig. 9 result shows that the PLNA787 of 3.2 μ M and 1.6 μ M can suppress the expression of ftsZ gene completely, and the PLNA787 of 0.8 μ M can partly suppress the expression of ftsZ gene, and naked LNA can not suppress the expression of ftsZ gene.
Sequence table
SEQUENCE LISTING
<110> the Fourth Military Medical University of P.L.A
Antisense antibacterial agent that <120> permeable membrane is peptide-mediated and its preparation method and application
<160>1
<170>PatentIn version 3.3
<210>1
<211>21
<212>DNA
<213> artificial sequence
<400>1
tgactcgcca ccagtaatat t 21
Claims (3)
1. the peptide-mediated antisense antibacterial agent of permeable membrane, is characterized in that: comprise (KFF)
3k-Cys-SMCC-C
6-LNA787, described (KFF)
3k-Cys-SMCC-C
6-LNA787 is permeable membrane peptide (KFF)
3between K and LNA787, be connected with halfcystine-(4-(N-maleimide methyl)-1-Cyclohexamide)-hexane;
The sequence of described LNA787 is 5'-T+*GA+CT*C+GCC*A+C+CA*GTAA*TA+T+*T-3', and wherein A is adenosine acid mono, and T is thymidylic acid monomer, and C is cytidine(C acid mono, and G is guanosine-acid mono; * X represents thio-modification, and X+ represents that LNA modifies, and X is A, T, C or G;
The Nucleotide of described thio-modification is as shown in formula I, and in the nucleotide structure that described LNA modifies, 2 '-O position and 4 '-C position forms Oxymethylene bridge by shrink effect, and connects into annular, as shown in formula II:
Described permeable membrane peptide (KFF)
3the sequence of K is 5'-KFFKFFKFFK-3', and wherein K is Methionin, and F is phenylalanine;
Described (KFF)
3k-Cys-SMCC-C
6the sequence of-LNA787 is:
5'-KFFKFFKFFK-Cys-SMCC-C
6-T+*GA+CT*C+GCC*A+C+CA*GTAA*TA+T+*T-3'；
Described antibacterial kind is methicillin-resistant staphylococcus aureus.
2. the peptide-mediated antisense antibacterial agent of permeable membrane according to claim 1, is characterized in that: the base in nucleotide monomer is guanine, VITAMIN B4, cytosine(Cyt), thymus pyrimidine.
3. a preparation method for the peptide-mediated antisense antibacterial agent of permeable membrane described in claim 1, is characterized in that: described (KFF)
3k-Cys-SMCC-C
6-LNA787 is by permeable membrane peptide (KFF)
3k and LNA787 are by the Cys-SMCC-C as spaced interval
6covalently bound, obtain with solid-phase synthesis preparation; Preparation process is as follows:
(1) permeable membrane peptide (KFF)
3the preparation of K: synthetic by fluorenylmethyloxycarbonyl solid phase synthesis process (Fmoc-SPPS), take HATU/DIEA as condensing agent, on MBHA-Rink resin, adopt Fmoc protection a-amino acid, the polypeptide that end (aminoterminal) solid phase synthesis C-end is halfcystine from C-end (carboxyl terminal) to N-, synthetic polypeptide is sheared and passes through RP-HPLC separation and purification through the TFA of high density; Permeable membrane peptide (KFF)
3the aminoacid sequence of K is KFFKFFKFFK, and K is Methionin (Lys), and F is phenylalanine (Phe);
(2) preparation of lock nucleic acid (LNA): adopt the solid phase phosphorous acid acid amides method of standard synthetic;
Concrete steps are: first by oligonucleotide chain 3 ' end nucleosides (N1) with its 3 '-OH by 1 long alkyl arm and the controlled micropore glass pearl of solid phase carrier (CPG) be coupled, the 5 '-OH of N1 protects with dimethoxytrityl (DMTr); Then start spreading oligonucleotide acid chain step by step from N1; 1 synthesis cycle comprises 4 steps; The first step: go protection, remove with trichoroacetic acid(TCA) (TCA) DMTr that CPG connects 5 on Nucleotide '-end, expose 5 '-OH, washing is in order to carrying out the next step; Second step: coupled reaction, before condensation, deoxynucleoside phosphoramidite monomer and tetrazolium are mixed and fed into synthetic post, tetrazolium provides a proton to the N atom of diisopropylamino on 3 ' phosphoric acid, protonated Diisopropylamine is a good free group, form this active intermediate of phosphoramidite tetrazolium with tetrazolium, this step tetrazolium is excessive has guaranteed monomer activation fully; When phosphoramidite tetrazolium contacts with CPG connected Nucleotide N1, there is nucleophilic reaction with its 5 '-OH, condensation occurs and take off tetrazolium, acetonitrile washing; The 3rd step: capping, add aceticanhydride and dimethyl aminopyridine, make not participate under the oligonucleotide chain acetyl of reaction, acetylizad oligonucleotide chain is not participated in next step reaction; The 4th step: oxidizing reaction, add iodine, make the phosphorous acid of trivalent change more stable pentavalent phosphoric acid into; After completing, above-mentioned circulation carries out second synthesis cycle; Every experience one is taken turns circulation, extends 1 Nucleotide; The oligonucleotide chain extending is fixed on insoluble solid phase carrier all the time, and excessive unreacted reactant or resolvent are by filtering or washing and remove; Synthetic and the aforesaid method difference of thio-modification Nucleotide have two: one, and vulcanization reaction replaces oxidizing reaction, and the 2nd, capping completes after vulcanization reaction; The synthetic method of the Nucleotide that LNA modifies is the same, and just nucleotide monomer is the nucleotide monomer of modifying through LNA, directly carries out above-mentioned four-step reaction;
Wherein, the sequence of LNA787 is 5'-T+*GA+CT*C+GCC*A+C+CA*GTAA*TA+T+*T-3', and wherein A is adenosine acid mono, and T is thymidylic acid monomer, and C is cytidine(C acid mono, and G is guanosine-acid mono; * X represents thio-modification, and X+ represents that LNA modifies, and X is A, T, C or G; The Nucleotide of thio-modification is as shown in formula I, in the nucleotide structure that LNA modifies, 2 '-O position and 4 '-C position forms Oxymethylene bridge by shrink effect, and connect into annular, this annular bridge has locked the configuration of furanose, reduce the snappiness of ribose structure, increase the stability of phosphate backbone local structure, as shown in formula II;
Adopt HPLC to carry out separation and purification to LNA crude product, obtain purity and be 98.58% LNA787, the molecular weight that MALDI-TOF measures is 6700.28;
(3) permeable membrane peptide (KFF)
3connection and the Purification of K and lock nucleic acid LNA: when oligonucleotide chain reaches after predetermined length, excessive SMCC joins on the oligonucleotide being connected on CPG, hatches 1h, fully washs final vacuum dry; Be connected to oligonucleotide on CPG and excessive permeable membrane peptide overnight incubation altogether, fully washing, (KFF)
3the connector of K and LNA cuts from solid phase carrier, and fresh strong aqua is sloughed protecting group, vacuum-drying; By (KFF)
3the connector crude product of K and LNA carries out purifying by HPLC, and identifies by MALDI-TOF mass spectrum; Permeable membrane peptide (KFF)
3the connector of K and LNA787 is by permeable membrane peptide (KFF)
3between K and LNA with halfcystine-(4-(N-maleimide methyl)-1-Cyclohexamide)-hexane (Cys-SMCC-C
6) connect wherein (KFF)
3k is positioned at halfcystine end, and LNA is positioned at hexane cardinal extremity, and concrete structure is as follows:
Adopt the crude product of the connector of RP-HPLC to permeable membrane peptide and LNA to carry out separation and purification, obtain purity and be 91.13% permeable membrane peptide (KFF)
3the connector of K and LNA787, i.e. the peptide-mediated antisense antibacterial agent of permeable membrane, the molecular weight that MALDI-TOF measures is 8616.14.
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| CN101346466A (en) * | 2005-12-22 | 2009-01-14 | 凯津公司 | Improved targeted nucleotide exchange with LNA modified oligonucleotides |
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| CN1387567A (en) * | 1998-11-11 | 2002-12-25 | 潘特科有限公司 | Conjugates between peptides and nucleic acid analog, such as PNA, LNA or morpholino |
| CN101346466A (en) * | 2005-12-22 | 2009-01-14 | 凯津公司 | Improved targeted nucleotide exchange with LNA modified oligonucleotides |
| CN101200483A (en) * | 2007-05-11 | 2008-06-18 | 中国人民解放军第四军医大学 | Antisense nucleic acid of resistant and tolerant dimethoxyphenecillin staphylococcus aureus drug resistant gene |
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