CN107573289A - A kind of compound of aldehyde radical uracil of specific chemical mark 5 and labeling method and application - Google Patents
A kind of compound of aldehyde radical uracil of specific chemical mark 5 and labeling method and application Download PDFInfo
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Abstract
The invention discloses a kind of compound of aldehyde radical uracil of specific chemical mark 5 and labeling method and application.By the compound 1 with azido group can selectivity and the aldehyde radicals of 5 aldehyde radical uracils react, add the biotin DBCO S S PEG with alkynyl3Biotin and nitrine occur not needing the click reactions of catalyst.Using the 5 aldehyde radical uracil specific chemical labeling methods of the present invention, the nucleic acid samples that specific enrichment contains 5 aldehyde radical uracils can be achieved, the sequence distributed intelligence of 5 aldehyde radical uracils in analyzing nucleic acid molecules.The present invention provides effective research method for epigenetics and nucleic acid chemistry biological study field.
Description
Technical field
The present invention relates to the chemical labeling of epigenetic modification base and detection method, more particularly to a kind of compound 1 and
Using the method for the compound specificity chemical labeling 5- aldehyde radical uracils, and its answering in mark, detection, sequencing etc.
With.
Background technology
In nature in addition to the presence of four kinds of natural bases, also in the presence of substantial amounts of modified base.Wherein DNA oxidative damages
Important products --- 5- aldehyde radicals uracil (hereinafter referred to as 5fU) is modification alkali naturally occurring in genome and less content
Base.And because it is in DNA reproduction process, mispairing can be produced, such as 5fU matches with guanine, and then cause gene to be dashed forward
Become, at the same be also induce a variety of diseases an important factor for one of.During the active demethylation of organism nucleic acid, inherently
Repaired along with the oxidative damage to modified base, excision.Univ Munich Germany Thomas professors Carell in 2014 are first
Report TET enzymes and can aoxidize thymidine and become 5-hydroxylmethyluracil.This result discloses the oxidation production of thymidine
Thing (5-hydroxylmethyluracil, 5- aldehyde radical uracils etc.) there may be certain epigenetics meaning.Cambridge is big within 2015
Learn Balasubramanian Shanker and screen a series of biotinylated organic molecule for 5- aldehyde radical uracils
Enrichment, and in 2017, be used in for Eukaryotic parasite Leishmania (eukaryote parasite
Leishmania the genome sequencing of 5hmU).5fU can produce via free-radical oxidation, or by Fenton effect, gamma
Ray and oxydasis produce.5fU can induce DNA that base mispairing occurs, and cause DNA structure to change, and occur with albumen
Interaction.It is widely present in vivo and content is seldom.
Comparision contents just because of 5fU are few, and there is presently no the technology that 5fU in full-length genome is sequenced.Therefore,
Need to develop a kind of new high selectivity, efficient 5fU tagging and testing methods, this is to being pushed further into epigenetic demethyl
Change research has the function that positive.
The content of the invention
In order to overcome the deficiencies in the prior art, it is an object of the invention to provide a kind of compound 1 and utilizationization
The method that the specific chemical of compound 1 marks 5- aldehyde radical uracils, the nucleic acid sample of the specific enrichment uracil of aldehyde radical containing 5- can be achieved
Product, the sequence distributed intelligence of 5- aldehyde radical uracils in analyzing nucleic acid molecules.
In order to realize the above object the present invention uses following technical scheme：
The first aspect of the present invention, there is provided a kind of compound 1, its structural formula is shown in formula I：
The second aspect of the present invention, there is provided a kind of method based on the specific chemical of compound 1 mark 5- aldehyde radical uracils,
Comprise the following steps：
(1) aldehyde radical of the compound 1 with azido group and 5- aldehyde radical uracils is subjected to dehydration condensation, generationization
Compound B, is shown below：
(2) with compound 2 click reactions occur for compound B, and the compound 2 is the biotin DBCO-S- with alkynyl
S-PEG3-biotin。
The third aspect of the present invention, there is provided one kind carries out 5- based on the specific chemical of compound 1 mark 5- aldehyde radicals uracil
The method of aldehyde radical uracil genome sequencing, comprises the following steps：
(1) genomic DNA is extracted, with the ultrasonic fragment for interrupting instrument and being broken into 250-400bp, a part is used as gene
Group DNA mark, another part give over to control group and directly build storehouse；
(2) genomic DNA that mark group interrupts adds compound 1 and handled, and compound 1 is carried out with 5fU on genome
Reaction；
(3) genomic DNA marked by compound 1 occur again with compound 2 click reaction, the compound 2 be with
The biotin DBCO-S-S-PEG of alkynyl3-biotin；
(4) with step (3) handle after DNA and modified Streptavidin magnetic bead be incubated after, collect containing biology
The chain of element, the i.e. DNA with 5fU；
(5) DNA for the mark group for obtaining control group and step (4) carries out library construction respectively, carries out the sequencing of two generations
(NGS), data analysis；Using control group data as background, data enrichment times are more than 4 times for effectively enrichment in mark group, with ginseng
Examine genome GRCm38.p5.genome.fa to be compared, obtain distribution of the fragment containing fU in mouse genome.
The mode for interrupting genomic DNA in above-mentioned steps (1) is：With Qubit Fluorometer by the gene of extraction
Group DNA is quantified, and is configured to 20 μ g/mL solution, is interrupted instrument with Covaris ultrasounds and interrupt genomic DNA, arrange parameter
It is as follows：Power is 175W, and ultrasonic time 420 seconds, control temperature is between 4 DEG C -16 DEG C；The DNA interrupted is coagulated with 1% agarose
Gel electrophoresis carry out the checking of fragment length.
The genomic DNA interrupted in step (2) adds the mode that compound 1 is handled：The gene interrupted to 20 μ g
5 μ L sodium acetate (NaOAc) buffer solution (1M, pH 5.0) is added in group DNA, 5 μ L compounds 1 is added and (250mM, uses DMSO
Dissolving), it is 50 μ L to add water to reaction system volume；System in oscillator 37 DEG C reaction 12 hours after with gel column remove it is unnecessary
Micromolecular compound, such as compound 1.
The 5fU being labeled in step (3) is again with the sides of click reactions occurs with alkynyl-modified biotin (biotin)
Formula is：5 μ L DBCO-S-S-PEG is added in the genomic DNA of purifying into step (2)3- biotin (20mM), system are being shaken
Swing in device and to remove unnecessary micromolecular compound such as DBCO-S-S-PEG with gel column after 37 DEG C of reactions 2 hours3-biotin。
The processing mode for transferring the genomic DNA with biotin in step (4) with magnetic bead is：By DNA after purification with
After magnetic bead with Streptavidin is incubated 15 minutes, in standing 5 minutes on magnetic frame, clear liquid, magnetic bead binding＆ are discarded
Washing buffer are washed 3 times, discard clear liquid, add 100 μ L (50mM) dithiothreitol (DTT) (DTT)；37 DEG C in oscillator
After reaction 2 hours, magnetic bead is fixed on magnetic frame 3 minutes, collects liquid, and product removes DTT with Purification Kit；Obtain
DNA quantified with Qubit Fluorometer.
The processing mode of genomic DNA chain progress library construction is in step (5)：The genome for taking 20ng magnetic beads to transfer
DNA is used for library construction, and control group is that the genomic DNA 20ng directly interrupted is used for library construction；Other operation according to
The specification that Rubicon Genomics Thruplex DNA-Seq kit build storehouse kit is carried out.
The principle of the present invention as shown in figure 1, the present invention is first to react the genomic DNA after ultrasonic interrupt with compound 1,
Click reactions occur with the biotin with alkynyl and the azido group of compound 1 again.Finally with the magnetic with Streptavidin
Pearl transfers the fragment containing 5fU.
The fourth aspect of the present invention, there is provided for detecting the kit of 5- aldehyde radical uracil bases, inclusion compound 1.
A further object of the present invention is to provide application of the compound 1 at following aspects：
(1) in sequencing analysis genome 5- aldehyde radical uracils sequence distributed intelligence；
(2) DNA molecular of the uracil base of aldehyde radical containing 5- is enriched with；
(3) kit of the distributed intelligence of 5- aldehyde radical uracil bases in detection genome DNA sample is prepared.
The present invention is the aldehyde radical generation with 5- aldehyde radical uracils that can be selective by the compound 1 with azido group
Reaction, and other bases discord compound 1 is reacted；Add the biotin with alkynyl and nitrine occurs not needing catalyst
Click reacts.Biotin fragment is connected with by being transferred with the magnetic bead that Streptavidin is modified, you can by fU fragment from complete
Taken out in genome.After storehouse kit structure library is built, the sequencing of two generations is carried out, is realized first from hippocampus of mice body
The distributed intelligence of the fragment containing 5fU is obtained in full-length genome.
Brief description of the drawings
Fig. 1 is the schematic flow sheet of fU in marker gene group of the present invention.
Fig. 2 is high performance liquid chromatography (HPLC) figure of compound 1 and the DNA reaction containing 5fU, in order to is confirmed
Compound 1 and DNA are reacted.
Fig. 3 is the gel electrophoresis figure after the DNA with different modifying base is labeled, in order to confirms compound
1 and the DNA with fU are reacted.
Fig. 4 is the distribution map for transferring fU fragments in obtained hippocampus of mice body genome in the present invention by compound 1.
Embodiment
By combination accompanying drawing described further below it will be further appreciated that the features and advantages of the invention.The implementation provided
Example is only explanation to the inventive method, remaining content without limiting the invention in any way announcement.
The raw material of the compound used in the specific embodiment of the invention is bought from Beijing Yi Nuokai Science and Technology Ltd.s.
【Embodiment 1】The building-up process and sign of compound 1
3,4- diaminobenzoic acids (3g, 19.7mmol) and ethyl cyanoacetate (7mL, 65.8mmol) are taken in 100mL circle
In the flask of bottom, stirring reaction 1h at 180 DEG C.After reaction terminates, reaction system is poured into 100mL ether and separates out solid, is subtracted
Pressure filters, and solid uses silica gel column chromatography, dichloromethane：Methanol：Acetic acid=100：1：0.1 elution, obtains compound as white solid A
(1g, 25%).1H NMR(400MHz,DMSO-d6) δ 12.92 (s, 1H), 8.15 (s, 1H), 7.83 (d, J=8.4Hz, 1H),
7.61 (d, J=7.7Hz, 1H), 4.46 (s, 2H) .HRMS (ESI+) C10H8N3O2 +[M+H]+calculated 202.06110,
found 202.06082。
Specific reaction scheme is as follows：
Take compound A (290mg, 1.4mmol), 3- azidopropylamines (720mg, 7.2mmol) and HATU (1.1g,
2.9mmol) in 25mL round-bottomed flasks, 10mL DMFs and 5 drop triethylamines, stirring reaction at 25 DEG C are added
4h.After reaction terminates, reaction system decompression rotation is evaporated, solid uses silica gel column chromatography, dichloromethane：Methanol=100：1
To 40：1 elution, obtains compound as white solid 1 (330mg, 80%).1H NMR(400MHz,DMSO-d6)δ12.86(s,1H),
8.53 (t, J=5.5Hz, 1H), 8.08 (s, 1H), 7.74 (dd, J=8.4,1.3Hz, 1H), 7.59 (d, J=8.2Hz, 1H),
4.44 (s, 2H), 3.43 (t, J=6.8Hz, 4H), 1.80 (p, J=6.8Hz, 2H)13C NMR(100MHz,DMSO-d6)δ
167.12,147.33,129.10,122.12,116.96,49.05,37.19,28.96,18.95.HRMS(ESI+)C13H14N7O+
[M+H]+calculated 284.12543,found 284.12497。
Specific reaction scheme is as follows：
【Embodiment 2】Compound 1 and 5fU monomer reactions route and sign
5- aldehyde radicals uracil (30mg, 0.12mmol) and compound 1 (33mg, 0.12mmol) are taken in 25mL round-bottomed flasks
In, add 10mL methanol and 5 drop acetic acid, stirring reaction 15h at 50 DEG C.After reaction terminates, reaction system decompression rotation is evaporated,
Solid uses silica gel column chromatography, dichloromethane：Methanol=50：1 to 15：1 elution, obtain yellow solid compound B (36mg,
60%).1H NMR(400MHz,DMSO-d6)δ13.39(s,1H),11.98(s,1H),8.95(s,1H),8.57(s,1H),
8.36-7.90 (m, 2H), 7.67 (dd, J=78.5,12.8Hz, 2H), 6.20 (t, J=6.6Hz, 1H), 5.35 (s, 1H),
4.99 (s, 1H), 4.35-4.25 (m, 1H), 3.90 (dd, J=6.8,4.1Hz, 1H), 3.63 (d, J=4.0Hz, 2H), 3.52-
3.40(m,4H),2.37–2.11(m,2H),1.90–1.72(m,2H).13C NMR(100MHz,DMSO-d6)δ167.04,
162.12,149.76,143.38,142.24,138.00,135.09,129.33,123.61,122.04,118.71,116.68,
111.55,107.86,100.39,88.68,86.20,71.21,61.86,49.06,40.78,37.22,28.94.HRMS(ESI
+)C23H24N9O6 +[M+H]+calculated 522.18441,found 522.18419。
Specific reaction scheme is as follows：
【Embodiment 3】The mark of mouse gene group DNA
The genomic DNA (gDNA) of extraction is dissolved in ultra-pure water after being interrupted with Ultrasound Instrument, and with Qubit Fluorometer
Quantitative instrument quantifies to gDNA.The mark of genomic DNA mainly divides 2 steps, as shown in figure 1, the first step：To 1.5mL's
20 μ L gDNA (1 μ g/ μ L) is added in EP pipes, 5 μ L sodium acetates buffer (pH 5.0), (250mM is dissolved in DMSO to 5 μ L compounds 1
In), 20 μ L ultra-pure water, it is put into after vibration centrifugation in oscillating reactions device, 37 DEG C are reacted 12 hours.After having reacted, gel column is used
Remove unnecessary compound 1.Second step：5 μ L DBCO-S-S-PEG is added in the product purified to the first step3- biotin, together
Sample, vibration remove unnecessary micromolecular compound with gel column after 37 DEG C of reactions being put into oscillator after mixing 2 hours.Finally
The mixture of purifying adds ultra-pure water polishing to 100 μ L volume.
【Embodiment 4】The enrichment of the fragment containing 5fU is transferred in mouse gene group DNA
The genomic DNA for the mark after purification that embodiment 3 obtains is transferred for magnetic bead, as shown in figure 1, detailed process is such as
Under：(1) after the magnetic bead with Streptavidin shakes up manually, 50 μ L magnetic bead is taken to rest on magnetic frame in 1.5mL EP pipes
Upper 3 minute, liquid is siphoned away with pipettor, magnetic bead washes 3 times (2) with 500 μ 1 × binding＆washing of L buffer and adds 50 μ
2 × binding＆washing of L buffer make magnetic bead scattered in the solution, are added into magnetic bead dispersion liquid in 50 μ L embodiments 1
Product after final purification, room temperature rotation incubation 15 minutes after being mixed with liquid-transfering gun piping and druming.(3) reaction tube is fixed on magnetic frame
On, siphon away liquid after standing 5 minutes.Washed 3 times with 500 μ 1 × binding＆washing of L buffer, in order to wash off
Biotin fragment is not contained.(4) DTT (50mM) of the 100 fresh configurations of μ L is added into magnetic bead, 37 DEG C of reactions in oscillator
2 hours.(5) reaction tube is fixed on magnetic frame 3 minutes, clear liquid is collected, with DNA Purification Kits.Obtained product is used
The structure of sequencing library is carried out after Qubit Fluorometer are quantitative.
【Embodiment 5】Control group and the structure in mark group library
The template that the genomic DNA for taking 20ng ultrasounds to interrupt is used as control group carries out library construction, takes 20ng mark purifying
The template that genomic DNA afterwards is used as mark group carries out library construction.Idiographic flow prepared by library is according to Thruplex DNA-
Seq kit (Rubicon Genomics) operating guidance is carried out.
【Embodiment 6】Two generation sequencing results are analyzed
DNA library prepared after by Suzhou Jin Weizhi companies carry out the sequencing of two generations, microarray dataset is Illumina Hiseq
150PE, pattern are pair end sequencing.Reference gene group file is GRCm38.p5.genome.fa (GENCODE numbers
Downloaded according to storehouse), data are analyzed using HOMER (v4.9) software.Using control group as background, wherein obtained in mark group
The effective rich segment of conduct of more than 4 times bigger than control group of fragment reads numbers.By reference to genome
GRCm38.p5.genome.fa is compared, and obtains distribution of the fragment containing fU in mouse genome.From Fig. 2 results
See, fU is mainly distributed in intergenic region and introne, and wherein intergenic region accounts for 62.12%, introne 3 6.02%, other areas
Domain such as extron, promoter, translational termination site, the region such as 5 '-UTR, 3 '-UTR account for 1.86% altogether.
【Embodiment 7】The analysis of high performance liquid chromatography (RP-HPLC)
In 1.5mL EP pipes, the DNA of 2 μ L (100 μM) containing 5fU bases, 5 μ L sodium acetate buffer (pH are added
5.0), 2 μ L compounds 1 (100mM is dissolved in DMSO), 38 μ L ultra-pure water, it is put into after vibration centrifugation in oscillating reactions device, 37 DEG C
Reaction 6 hours.After having reacted, unnecessary compound 1 is removed with gel column.The RP-HPLC tracers of the product of reaction, collect
260nm signal, by retention time it can be seen that compound 1 on the DNA fragmentation pass flag containing fU, as shown in Figure 3.
【Embodiment 8】The analysis of reaction selectivity
(ODN-T, ODN-5hmU, ODN-5fU, ODN-5hmC, ODN-5fC, ODN-AP) DNA with different modifying
(ODN-T, ODN-5fU are synthesized by Wuhan Jim Carrey company, and other DNA are synthesized by Dalian treasured biotech firm, the DNA of different modifying
Sequence be shown in Table 1) according to the reactions steps in embodiment 3 respectively with after the reaction purification of compound 1, every kind of DNA and compound 1 are anti-
Product that should after purification is analyzed by 20% denaturing gel electrophoresis, and because fU fragment is marked by compound 1, molecular weight increase is led
Electrophoretic velocity is caused to slow down.It can be seen that a new band on gel electrophoresis figure as shown in Figure 4.
DNA of the table 1 with different modifying sequence
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (6)
1. a kind of compound 1, it is characterised in that its structural formula is shown in formula I：
2. a kind of method of the specific chemical of the compound 1 mark 5- aldehyde radical uracils based on described in claim 1, its feature exist
In comprising the following steps：
(1) aldehyde radical of the compound 1 and 5- aldehyde radical uracils with azido group is reacted, generates compound B, it is specific anti-
Answer route as follows：
(2) with compound 2 click reactions occur for compound B, and the compound 2 is the biotin DBCO-S-S- with alkynyl
PEG3-biotin。
It is phonetic that 3. a kind of specific chemical of compound 1 mark 5- aldehyde radicals uracil based on described in claim 1 carries out 5- aldehyde radicals urine
The method of pyridine genome sequencing, it is characterised in that comprise the following steps：
(1) genomic DNA is extracted, with the ultrasonic fragment for interrupting instrument and being broken into 250-400bp, a part is used as genome
DNA mark, another part give over to control group and directly build storehouse；
(2) genomic DNA that mark group interrupts adds compound 1 and handled, and compound 1 is reacted with 5fU on genome；
(3) click reactions occur with compound 2 again for the genomic DNA marked by compound 1, and the compound 2 is with alkynyl
Biotin DBCO-S-S-PEG3-biotin；
(4) with step (3) handle after DNA and modified Streptavidin magnetic bead be incubated after, collect containing biotin
Chain, the i.e. DNA with 5fU；
(5) DNA for the mark group for obtaining control group and step (4) carries out library construction respectively, carries out the sequencing of two generations
(NGS), data analysis；Using control group data as background, data enrichment times are more than 4 times for effectively enrichment in mark group, with ginseng
Examine genome GRCm38.p5.genome.fa to be compared, obtain distribution of the fragment containing fU in mouse genome.
4. according to the method for claim 3, it is characterised in that
The mode for interrupting genomic DNA in step (1) is：The genomic DNA of extraction is carried out with Qubit Fluorometer
It is quantitative, 20 μ g/mL solution is configured to, instrument is interrupted with Covaris ultrasounds and interrupts genomic DNA, arrange parameter is as follows：Power
For 175W, ultrasonic time 420 seconds, control temperature is between 4 DEG C -16 DEG C；The DNA interrupted is entered with 1% agarose gel electrophoresis
The checking of row fragment length；
The genomic DNA interrupted in step (2) adds the mode that compound 1 is handled：The base interrupted to 20 μ g mark groups
Because adding 5 μ L sodium acetate (NaOAc) buffer solution (1M, pH 5.0) in group DNA, add 5 μ L compounds 1 and (250mM, use
DMSO dissolves), it is 50 μ L to add water to reaction system volume；System in oscillator 37 DEG C reaction 12 hours after removed with gel column
Unnecessary micromolecular compound, wherein including unnecessary compound 1；
The 5fU being labeled in step (3) is again with the modes of click reactions occurs with alkynyl-modified biotin (biotin)
For：5 μ L DBCO-S-S-PEG is added in the genomic DNA of purifying into step (2)3- biotin (20mM), system are vibrating
Unnecessary micromolecular compound is removed with gel column after 37 DEG C of reactions 2 hours in device, wherein including unnecessary DBCO-S-S-PEG3-
biotin；
The processing mode for transferring the genomic DNA with biotin in step (4) with magnetic bead is：By DNA after purification with carrying
After the magnetic bead of Streptavidin is incubated 15 minutes, in standing 5 minutes on magnetic frame, clear liquid, magnetic bead binding＆ are discarded
Washing buffer are washed 3 times, discard clear liquid, add 100 μ L (50mM) dithiothreitol (DTT) (DTT)；37 DEG C in oscillator
After reaction 2 hours, magnetic bead is fixed on magnetic frame 3 minutes, collects liquid, and product removes DTT with Purification Kit；Obtain
DNA quantified with Qubit Fluorometer；
The processing mode of genomic DNA chain progress library construction is in step (5)：The genomic DNA for taking 20ng magnetic beads to transfer is used
In library construction, control group is that the genomic DNA 20ng directly interrupted is used for library construction；Other operations are according to Rubicon
The specification that Genomics Thruplex DNA-Seq kit build storehouse kit is carried out.
A kind of 5. kit of detection 5- aldehyde radical uracil bases, it is characterised in that inclusion compound 1.
6. compound 1 described in claim 1 is in the application of following aspects：
(1) in sequencing analysis genome 5- aldehyde radical uracils sequence distributed intelligence；
(2) DNA molecular of the uracil base of aldehyde radical containing 5- is enriched with；
(3) kit of the distributed intelligence of 5- aldehyde radical uracil bases in detection genome DNA sample is prepared.
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| CN111041023A (en) * | 2018-10-11 | 2020-04-21 | 中国科学院天津工业生物技术研究所 | Specific nucleic acid binding proteins and methods for enriching for specific nucleic acids |
| CN111088252A (en) * | 2019-12-30 | 2020-05-01 | 西安交通大学 | Specific labeling method of 5-hydroxymethyl uracil on DNA |
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| CN114106012A (en) * | 2021-12-13 | 2022-03-01 | 宜昌博仁凯润药业有限公司 | Compound Biotin-PEG3-SS-DBCO, preparation method and application thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108530483A (en) * | 2018-01-15 | 2018-09-14 | 四川大学 | Wittig reagents based on coumarin skeleton and its preparation method and application |
| CN108530483B (en) * | 2018-01-15 | 2020-07-28 | 四川大学 | Wittig reagent based on coumarin skeleton and preparation method and application thereof |
| CN111041023A (en) * | 2018-10-11 | 2020-04-21 | 中国科学院天津工业生物技术研究所 | Specific nucleic acid binding proteins and methods for enriching for specific nucleic acids |
| WO2020155742A1 (en) * | 2019-01-28 | 2020-08-06 | 四川大学 | Method for deriving aldehyde-based pyrimidine, method for detecting 5-aldehyde cytosine, and use of aldehyde-based pyrimidine derivative |
| CN111088252A (en) * | 2019-12-30 | 2020-05-01 | 西安交通大学 | Specific labeling method of 5-hydroxymethyl uracil on DNA |
| CN111088252B (en) * | 2019-12-30 | 2021-07-13 | 西安交通大学 | Specific labeling method of 5-hydroxymethyl uracil on DNA |
| CN114106012A (en) * | 2021-12-13 | 2022-03-01 | 宜昌博仁凯润药业有限公司 | Compound Biotin-PEG3-SS-DBCO, preparation method and application thereof |
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