CN103382212A - 5'-bit modified puromycin compound, and preparation method and applications thereof - Google Patents
5'-bit modified puromycin compound, and preparation method and applications thereof Download PDFInfo
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- CN103382212A CN103382212A CN2013102822773A CN201310282277A CN103382212A CN 103382212 A CN103382212 A CN 103382212A CN 2013102822773 A CN2013102822773 A CN 2013102822773A CN 201310282277 A CN201310282277 A CN 201310282277A CN 103382212 A CN103382212 A CN 103382212A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to Chemical Invention technical field, it is related to a kind of 5' modified purine mycin compound, synthetic method and its purposes in drug screening. The structural formula of 5' modified purine mycin compound of the present invention is as follows:
Wherein: R1 is blocking group Fmoc, Boc or Cbz, and R2 is blocking group Ac, Bz or Pv. The compounds of this invention can be connected to the end 5' of oligonucleotide chain by solid phase phosphoramidite triester method, and for the screening of polypeptide drug, preparation method is easy, and combined coefficient is high, be easy to business and be combined to.
Description
Technical field
The invention belongs to chemical invention technical field, be specifically related to 5' position modification tetracycline compound and its production and use.
Background technology
In recent years, the in-vitro screening technology of protein has obtained development fast, this shows (Plasmic display) and cell display (Cell display) comprising early stage phage display (Phage display), plasmid, but all relate to cell transfecting because phage display technology is shown in when building the library and screening, the library capacity is subjected to the impact of transfection efficiency to be limited in 10
9～10
10, reduced library screening efficient, in addition, in a single day phage display library builds up, and is difficult to carry out effective external sudden change and restructuring again, and then has limited the diversity of molecular genetic in the library.Under this background, the J.W.Szostak of U.S. Harvard Medical School has set up the protein triage techniques that a kind of mRNA of being called as shows, this technology is mainly to have utilized tetracycline when expressing can enter ribosomal A site and newly-generated this characteristic of polypeptide formation covalent linkage, express external after the 3' end is connected with mRNA with the DNA of tetracycline, be built into the polypeptide libraries that can be used in the protein medicaments screening.But this method is take RNA as template, and formed RNA-protein fusion body is difficult to withstand harsh screening requirement, particularly easily is subject to the degraded of RNA enzyme, thereby affects whole screening process.
The DNA display technique that we propose be a kind of can be at the external Novel screen choosing method that carries out, except not being subjected to the cell transfecting effectiveness affects, its formed DNA-protein blend zoarium is also more stable.Therefore, DNA shows as a kind of emerging protein triage techniques, will be at new drug development, and the aspects such as protein interaction and proteomics demonstrate application space more widely.The realization of DNA display technique need to be connected to tetracycline the 5' end of DNA, and existing method can only be connected to tetracycline the 3' end of DNA.
Summary of the invention
One of purpose of the present invention is to provide 5' position modification tetracycline compound.
Two of purpose of the present invention is to provide the synthetic method of 5' position modification tetracycline compound.
Three of purpose of the present invention is to provide the purposes of 5' position modification tetracycline compound.
The objective of the invention is to be achieved through the following technical solutions:
5' of the present invention position modification tetracycline compound, its structural formula is as follows:
Advantage of the present invention: the compounds of this invention can be coupled to the 5'-end of DNA, is built into more stable protein medicaments screening system, and this compound prepares easyly, efficient, is easy to commercialization.
Below in conjunction with embodiment, the present invention is carried out concrete description.As known by the technical knowledge, the present invention can realize by other the embodiment that does not break away from its spiritual essence or essential feature.Therefore, following embodiment with regard to each side, all just illustrates, and is not only.All within the scope of the present invention or the change that is equal in scope of the present invention all be included in the invention.
Description of drawings
Fig. 1 is the schematic flow sheet of specific embodiment 1 tetracycline chemically modified.
Fig. 2 is the schematic diagram of specific embodiment 2 tetracyclines and oligonucleotide linked reaction.
Fig. 3 is the schematic flow sheet of specific embodiment 3 in-vitro screening albumen.
Fig. 4 is the expression of results figure of specific embodiment 4.
Embodiment
Below in conjunction with accompanying drawing, further illustrate the present invention by example.One skilled in the art will understand that these examples only are used for explanation the present invention, limit the scope of the invention and be not used in.
Embodiment 1(synthesis step)
The chemical modification reaction step of tetracycline is referring to Fig. 2.Concrete operation step is as follows:
a. take the 100mg tetracycline and be placed in eggplant-shape bottle, the acetonitrile that adds the new distillation of 2ml, use ultrasonic echography half an hour, resulting solution is placed in the ice-water bath stirring is cooled to 0 ℃, slowly add the 60ul triethylamine under nitrogen protection, after the solution clarification, 72mg fluorenes methoxy carbonyl acyl succinimide (Fmoc-Osu) is dissolved in the 1ml acetonitrile, 0 ℃ drops in above-mentioned reaction solution, continue to stir, after 30min, solution becomes white opacity liquid, sluggish is risen to room temperature and stirs 1h, until the TLC monitoring reaction is completed, filter with Büchner funnel and obtain the white solid crude product, resulting crude product is further purified by silica gel column chromatography and obtains P1, eluent methylene dichloride: methyl alcohol=8:1.
B. 150mg product P 1 is placed in eggplant-shape bottle; the pyrido that adds 2ml to process through distillation fully stirs evenly with stirrer; add the 200ul triethylamine at 0 ℃; after 15min, 400mg DMTrCl is dissolved in the 2ml pyridine; slowly join reaction solution under nitrogen protection in; be stirred to room temperature, until the TLC monitoring reaction is completed.Resulting reaction solution is mixed sample by concentrating under reduced pressure, dry method, obtain product P 2 by silica gel column chromatography, eluent is sherwood oil: ethyl acetate=1:3.(when TLC analyzes, product point owing to containing the DMT group, can be analyzed by baking sheet, can become orange with the product of DMT through baking sheet, and in sample, residual pyridine passes through high temperature and can spread apart, and so just product and other impure point can be distinguished mutually)
C. 420mg P2 is dissolved in the pyridine that 6ml processes through distillation; add 10mg4-Dimethylamino pyridine (DMAP); ice-water bath is cooled to 0 ℃; slowly add the 0.6ml aceticanhydride under nitrogen protection; after 3h, the TLC monitoring reaction is completed, and adds methyl alcohol cancellation reaction at 0 ℃; obtain product P 3 by silica gel column chromatography, eluent is sherwood oil: ethyl acetate=1:3.
D. 380mg P3 is dissolved in through anhydrous CaCl2 drying treatment and distilled methylene dichloride (5ml), ice-water bath is cooled to 0 ℃, add the 0.2ml dichloro acetic acid, 0 ℃ is stirred to room temperature, finish after reaction 2h, this operation is comparatively simple, and reaction finishes to obtain product P 4 by the dry method loading by silica gel column chromatography, and eluent is sherwood oil: ethyl acetate=1:3.
E. claim 30mg P4; under the N2 protection; add the 250ul pyridine; 35 ℃ are stirred 15min P4 are dissolved fully; after dissolving, solution is faint yellow; reaction solution is cooled to 0 ℃ to be continued to stir; and add the inferior phosphoryl chloride of 35ul DIPEA and 20ul; after 1h; reaction is warming up to 15 ℃ continues reaction 4h, TLC plate monitoring reaction is completed, and obtains product P 5 through the alkaline silica gel column purification; the preparation process of its neutral and alkali post is as follows: the mixed solution of getting 200ml triethylamine: methylene dichloride=3:97 is washed into alkalescence with pillar, then washes pillar to washing out with the 100ml methylene dichloride.
Table 1: the yield of representative compound and mass-spectrometric data
Embodiment 2, Fig. 2 are that the tetracycline after modification is connected with the DNA of one section fixed sequence program, and reactions steps and present widely used solid phase phosphoramidite triester method are basic identical, and coupled product obtains by the high performance liquid phase separation and purification.As shown in FIG.: contain the polyoxyethylene glycol of 12 carbon near the Spacer18 representative of DNA5' end, the rC representative base that the 5'-3' direction is turned round on oligonucleotide chain.
Embodiment 3, carry out the in-vitro screening of polypeptide by 5' position modification tetracycline
The flow process of in-vitro screening albumen is seen Fig. 3.
(1) build the double-stranded DNA library that comprises stochastic sequence.Synthesize single-stranded DNA banks with stochastic sequence by chemical process, add the pcr amplification that isocyatic downstream primer carries out two circulations, the final double-stranded DNA random library that obtains to comprise T7 promotor, enhanser, initiator codon, stochastic sequence, affinity purification label coding sequence.
The single stranded DNA stochastic sequence:
TAATACGACTCACTATAGGAGGACGAAATG(NNN)
9CACCACCACCATCATCATCAGC TGCGTAACTC
Downstream primer: GAG TTA CGC AGC TGA TGA
Reaction system and PCR condition:
The PCR condition is: 95 ℃ of preheating 1min; 95 ℃ of sex change 30s, 45 ℃ of renaturation 45s, 72 ℃ are extended 45s, 2 circulations.
(2) in-vitro transcription.Take double-stranded DNA as template, to transcribe under the polysaccharase effect and obtain mRNA, reaction system is as follows:
(3) vivoexpression of polypeptide.Carry out take mRNA as template first making itself and end with the oligonucleotide chain annealing complementation of tetracycline before the polypeptide translation, add rabbit reticulocyte lysate to express, reaction adds Repone K, magnesium chloride to make K after finishing
+, Mg
2+Ionic concn reaches respectively 500mM and 50mM and places 50min in room temperature.Take a small amount of expression system as example, whole reactions steps and system are as follows:
Catch primer (5' end band tetracycline, 10uM) 1.7ul
MRNA (approximately 10uM) 1.7ul
ddH
2O 7.6ul
(67 ℃ of heating 10min, room temperature is placed 5min)
Aminoacid mixture 1 (not containing methionine(Met)) 0.5ul
Aminoacid mixture 2 (not containing leucine) 0.5ul
Rabbit reticulocyte lysate 8.5ul
(30 ℃ of reaction 20min place 40min on ice)
KCl (3M) 6.3ul (final concentration 500mM)
MgCl
2(0.5M) 3.8ul (final concentration 50mM)
(room temperature is placed 50min)
When carrying out great expression, reaction system is amplified in proportion as required and is got final product.
Embodiment 4, Fig. 4 carry out the electrophoresis result that expression of polypeptides is processed with Proteinase K later on.At first use one section of isotropic substance 32P mark with the DNA of mRNA template complementary sequence, and be connected with coupled product in embodiment 3 under the existence of clamping plate, obtain can be used for catching the band tetracycline primer Pu16-L1 of polypeptide, then make the expression of carrying out polypeptide after itself and the complementation of mRNA template annealing, obtain DNA-polypeptide syzygy (swimming lane 2 top bands), this syzygy be degraded (swimming lane 4) after Proteinase K is processed.Swimming lane 1 is to catch the not expression of results of purine-containing mycin of primer.Swimming lane 3 is that mRNA shows experiment, is used as the positive control of vivoexpression, the mRNA-polypeptide syzygy that forms in expression (swimming lane 3 top bands) can degrade equally after Proteinase K is processed (swimming lane 5).
Claims (5)
1. 5' position modification tetracycline compound, its structural formula is as follows:
Wherein: R
1Be blocking group Fmoc, Boc or Cbz, R
2Be blocking group Ac, Bz or Pv.
2. 5' position modification tetracycline compound according to claim 1, is characterized in that: can be connected to phosphodiester bond the 5' end of oligonucleotide chain.
3. a method for preparing 5' claimed in claim 1 position modification tetracycline compound is: a. is in organic solvent, add respectively fluorenes methoxy carbonyl acyl succinimide and natural tetracycline to react, obtain amino protected preliminary modification tetracycline product; B. add the dimethoxytrityl methyl chloride to react with it, obtain the tetracycline that the 5' position is protected by dimethoxytrityl; C. add aceticanhydride and its reaction, the modification tetracycline that acquisition 2' is protected by ethanoyl the position; D. add dichloro acetic acid or trichoroacetic acid(TCA) to slough the dimethoxytrityl group of 5' position, obtaining the 5' position is the modification tetracycline of hydroxyl; E. add inferior phosphoryl chloride to react with it, obtaining the 5' position is the modification tetracycline target product of phosphoramidite protection.
4. tetracycline compound according to claim 1, it is characterized in that: this compound is a kind of protein synthesis inhibitor, the amino-terminal end gene that in its structure and aminoacyl-tRNA molecule, adenosine is connected is similar, can enter ribosomal A site and form covalent structure with the polypeptide chain that is extending when expressing.
5. 5' position modification tetracycline compound claimed in claim 1, be used for the screening of polypeptide drug.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| WO2013019794A1 (en) * | 2011-08-01 | 2013-02-07 | The General Hospital Corporation | Protein and peptide libraries |
| US20130122535A1 (en) * | 2011-11-10 | 2013-05-16 | President And Fellows Of Harvard College | Methods and compositions for labeling polypeptides |
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2013
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| US5824793A (en) * | 1996-02-21 | 1998-10-20 | Lynx Therapeutics, Inc. | Solid phase synthesis of oligonucleotide N3'-P5' phosphoramidates |
| CN1373768A (en) * | 1999-09-10 | 2002-10-09 | 杰龙公司 | Oligonucleotide N3'-P5' thiophosphoramidates, their synthesis and use |
| US20090076295A1 (en) * | 2000-09-11 | 2009-03-19 | Affymetrix, Inc. | Photocleavable protecting groups |
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| CN103382212B (en) | 2016-08-31 |
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