CN110331447B - Method for preparing On-DNA aryl alkyne compound in construction of DNA coding compound library - Google Patents
Method for preparing On-DNA aryl alkyne compound in construction of DNA coding compound library Download PDFInfo
- Publication number
- CN110331447B CN110331447B CN201910609568.6A CN201910609568A CN110331447B CN 110331447 B CN110331447 B CN 110331447B CN 201910609568 A CN201910609568 A CN 201910609568A CN 110331447 B CN110331447 B CN 110331447B
- Authority
- CN
- China
- Prior art keywords
- equivalents
- palladium
- bis
- dna
- hours
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B40/00—Libraries per se, e.g. arrays, mixtures
- C40B40/04—Libraries containing only organic compounds
- C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/08—Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
- C40B50/10—Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support involving encoding steps
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Structural Engineering (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention discloses a method for preparing an On-DNA aryl alkyne compound in the construction of a DNA coding compound library, which comprises the following steps: the On-DNA aryl alkyne compound is prepared by taking an On-DNA terminal alkyne compound as a substrate and carrying out Sonogashira coupling reaction with a small-molecular aryl halide reagent in the presence of a palladium catalyst, a ligand and alkali. The reaction method provided by the invention provides a new method for the cap reaction of the On-DNA terminal alkyne compound, can obviously increase the diversity of the DNA coding compound library of the On-DNA terminal alkyne compound, has high yield, wide substrate universality, mild conditions and convenient operation, and is suitable for the synthesis of the DNA coding compound library by a porous plate.
Description
Technical Field
The invention belongs to the technical field of DNA coding compound libraries, and particularly relates to a method for obtaining an On-DNA aryl alkyne compound by reacting an On-DNA terminal alkyne compound with a small-molecular aryl halide reagent in the construction of a DNA coding compound library.
Background
The teaching of Sydney Brenner and Richard Lerner of the research institute of Scripps, USA, in 1992, proposed the concept of DNA Encoded Library of compounds (DEL) (reference: Proc. Natl. Acad. Sci.,1992,89,5381, U.S. Pat. No. 4,5573905) by linking an organic small molecule agent to a unique sequence of DNA at the molecular level (i.e., DNA labeling of small molecule agents), rapidly constructing a large Library of compounds by two to many cycles using the "combinatorial-resolution" strategy of combinatorial chemistry, in which each compound in the Library consists of residues of a different small organic molecule agent and is labeled with DNA of the corresponding unique base sequence, affinity-screening a very small Library of DNA-encoding compounds with a target, washing away the Library molecules of compounds that are not adsorbed to the target, washing away the remaining Library molecules of compounds that are adsorbed to the target, the concentration of the molecules of the obtained compound library is low, and the molecules are difficult to analyze and identify by a conventional method, but the DNA part in the obtained compound library molecules adsorbed to a target can be copied and amplified by a Polymerase Chain Reaction (PCR) unique to DNA until the obtained DNA quantity can be identified by a DNA sequencer, the sequenced data is decoded by a relation table between an organic small molecule reagent and each specific DNA base sequence which are created when a DNA coding compound library is constructed, so that the organic small molecule reagent corresponding to a specific compound capable of identifying the molecules with potential activity is found, the organic small molecule reagents are combined together by a traditional organic synthesis method to obtain a screened target molecule, and the physiological activity of the target molecule is detected and confirmed.
The method for constructing DNA coding compound Library mainly includes three kinds, the first kind is DNA-guided Chemical Library Synthesis (DTCL) mainly obtained by using DNA template technology from Ensemble corporation in America, the second kind is DNA-Recorded Chemical Library (DRCL) mainly obtained by using DNA marking technology from GSK corporation in America, X-Chem corporation and domestic leader corporation, the third kind is coding Self-assembly molecule Library (ESAC) mainly obtained by Fragment-based drug design (Fragment-based drug discovery, FBDD) technology from Philogen corporation in Switzerland, the method for constructing DNA coding compound Library which is industrially applied in large quantity at present is mainly the second kind, the method is simple to operate and lower in cost, and can quickly obtain a DNA coding compound library containing a large number of compounds by using a combinatorial chemical method.
In addition to the DNA starting fragment (see the present invention patents: CN201711263372.3, CN201711318894.9), a large number of DNA tags and small organic reagents are required which can be reacted in a certain order. The DNA tag code can be obtained by a computer program (see the present invention patent: CN201711247220.4), and a specific DNA sequence primer can be obtained by a DNA synthesizer. The small organic molecule reagent can be obtained by screening the obtained reagent list by using a certain computer program (see the invention patent of the company: CN 201810378969.0).
One of the major efforts in the DEL library field at present is the development of chemical reactions On DNA, referred to as On-DNA chemistry. Because DNA must be kept stable in a certain water phase, pH, temperature, metal ion concentration and inorganic salt concentration, the On-DNA chemical reaction with small DNA damage, better recovery rate and wide substrate adaptability is required for large-scale application in the synthesis of DNA coding compound libraries. There are more than 50 kinds of On-DNA chemical reactions reported in the prior art, each reaction condition is one kind or more than ten kinds, so that under the same other conditions, the more the kinds of On-DNA chemical reactions are, the more the conditions are, the more the selectivity is in designing the DNA coding compound library, the higher the synthesis success rate of the final library is, and the more the diversity of the obtained library is.
TABLE 1 On-DNA chemistry reaction species and specific conditions that can be used for DRCL construction
In the On-DNA chemical reaction, the selection of buffer solution is very important, and we have determined several commonly used buffer solution preparation and quality inspection methods (see the invention patent of the company: CN201811181396.9 for details) and provided several specific On-DNA chemical reactions under the condition of the buffer solution (see the invention patent of the company: CN201811181396.9, CN201811546746.7 for details).
The bond-forming chemical reaction most commonly used in the construction of libraries of DNA-encoding compounds today is: amide bond formation reaction, capping reaction of other amines (reductive amination, substitution, (thio) urea formation, carbamate formation, sulfonylation, etc.), Suzuki coupling reaction, Sonogashira coupling reaction, Heck coupling reaction, Buchwald coupling reaction, Ullmann coupling reaction, etc. (reference: angew. chem. int. ed.,2019,58, 10.1002/anie.201902489).
At present, most of On-DNA metal-catalyzed coupling reactions take On-DNA aryl halides as substrates, the application of the metal-catalyzed coupling reactions in a DNA coding compound library is limited, the reported On-DNA Sonogashira coupling reactions mainly comprise three methods, and Satz et al of Roche company reports that Pd (OAc) is used in 20152The Sonogashira coupling reaction in which TPPTS and pyrrolidine catalyze the reaction of On-DNA aryl iodide with a small-molecule terminal alkyne reagent in an aqueous phase at 65 ℃ (reference: Bioconjugate chem.,2015,26,1623-1632), and the Arico-Muendel et al by GSK introduced Pd (PPh) by GSK in a review in 20163)4And the Sonogashira coupling reaction in which pyrrolidine catalyzes the reaction of On-DNA aryl iodide with a terminal alkyne reagent generated in situ from an aldehyde by Seyferth-Gilbert recarburization in the aqueous phase at 65 ℃ (reference: Med. chem. Commun.,2016,1898-1909), Neri et al, of the Suzurich Federal institute of technology (ETH Surich) reported the use of [ PdCl (allyl) ]2TPPS and sodium carbonate catalyze Sonogashira coupling reaction of On-DNA aryl iodide and terminal alkyne reagent at 70 ℃ in water phase (reference: Helv. Chim. acta,2019,102, e 1900033).
Because On-DNA aryl iodide is mainly composed of DNA-NH in the synthesis of DNA coding compound library2And small molecular carboxylic acid aryl iodine binary compound through condensation reaction, wherein the three reaction methods are all obtained throughThe Sonogashira coupling reaction of the On-DNA aryl iodide and the micromolecule terminal alkyne reaction has the defects of few types of commercially available terminal alkyne reagents, difficult solution storage and the like, and the type and the number of micromolecule carboxylic acid aryl iodide binary compounds limit the application of the Sonogashira coupling reaction in the synthesis of DNA coding compound libraries.
In order to solve the problems, a method which is simple, convenient and fast and can convert an On-DNA terminal alkyne compound and a small-molecular aryl iodide or bromide reagent into an On-DNA aryl alkyne compound through a Sonogashira coupling reaction is hoped to be developed, so that the application range of the Sonogashira coupling reaction in the synthesis of a DNA coding compound library can be expanded.
Disclosure of Invention
The invention aims to solve one of the technical problems and provides a Sonogashira coupling reaction which is used for constructing a DNA coding compound library, takes part in a small-molecular aryl halide reagent Ar-X and takes an On-DNA terminal alkyne compound as a substrate. Wherein the structural formula of the On-DNA terminal alkyne compound is as follows: 
The structural formula of the prepared On-DNA aryl alkyne compound is shown as
Wherein, the DNA in the structural formula is a single-stranded or double-stranded nucleotide chain obtained by polymerizing artificially modified and/or unmodified nucleotide monomers;
ar group is the residual part of small molecule aryl halide reagent with molecular weight below 1000 which can participate in the reaction on the product, and X is bromine or iodine.
The second technical problem to be solved by the invention is to provide a method for converting an On-DNA terminal alkyne compound into an On-DNA aryl alkyne compound through a Sonogashira coupling reaction.
The method is characterized in that an On-DNA terminal alkyne compound is used as a raw material, any one or more of acetonitrile, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, methanol, ethanol, tert-butyl alcohol, isopropanol, tetrahydrofuran, 1, 4-dioxane, water, an inorganic salt buffer solution, an organic acid buffer solution and an organic base buffer solution is used as a solvent, and the On-DNA terminal alkyne compound reacts with a small-molecular aryl halide reagent at the temperature of 20-100 ℃ for 0.5-24 hours in the presence of a palladium catalyst, alkali or a ligand, and the specific reaction equation is as follows:
the palladium catalyst is palladium chloride, palladium bromide, palladium iodide, bis (acetonitrile) palladium dichloride, ethylenediamine palladium chloride, palladium trifluoroacetate, palladium acetate, palladium sulfate, palladium nitrate, palladium dipropionate, palladium acetylacetonate, hexafluoroacetylacetonatopalladium, dichloro (norbornadiene) palladium, (1, 5-cyclooctadiene) palladium dichloride, dichloro (N, N, N-tetramethylethylenediamine) palladium, dichloro (1, 10-phenanthroline) palladium, bis (dibenzylideneacetone) palladium, tris (dibenzylideneacetone) dipalladium, tetrakis (acetonitrile) bis (trifluoromethanesulfonic) palladium, tetrakis (triphenylphosphine) palladium, tetrakis (tri-o-tolylphosphine) palladium, triphenylphosphine palladium acetate, bis (tri-o-tolylphosphine) palladium, bis [1, 2-bis (diphenylphosphino) ethane ] palladium, bis (tri-t-butylphosphino) palladium, dihydrodichloro bis (di-t-butylhydroxyphosphinylidene) palladium, bis (di-t-butylphosphinidene) palladium, palladium trifluoroacetyl) palladium, palladium acetate, bis (tris-o-tolylphosphine) palladium, bis (1, 2-bis (dibenzylideneacetophenone) palladium, bis (tri-t-butylphosphino) palladium, bis (tris-butylidene) palladium, bis (tert-butylidene) palladium, bis (tris-tetramethylammonium) palladium, bis (tert-butylidene) palladium, bis (iodonium), palladium, bis (methyl) palladium, bis (ethyl, bis (methyl) palladium, bis (ethyl, bis (methyl) ethyl, bis (ethyl, and (ethyl, palladium, one or a mixture of more of bis (triphenylphosphine) palladium dichloride, (1, 3-bis (diphenylphosphino) propane) palladium chloride, [1,1 '-bis (diphenylphosphino) ferrocene ] palladium dichloride dichloromethane complex, dibromo bis (tri-tert-butylphosphino) dipalladium, 1' -bis (di-tert-butylphosphino) ferrocene palladium dichloride and bis (tricyclohexylphosphine) palladium dichloride; preferably, the palladium catalyst is tetrakis (triphenylphosphine) palladium.
The ligand is triphenylphosphine, tris [3, 4-bis (trifluoromethyl) phenyl ] phosphine, tris (4-fluorophenyl) phosphine, tris (3-bromophenyl) phosphine, tris (2-methoxyphenyl) phosphine, tris (4-methoxy-3, 5-dimethylphenyl) phosphine, tris (4-dimethylaminophenyl) phosphine, tris (4-chlorophenyl) phosphine, tris (3-chlorophenyl) phosphine, triphenylphosphine-3, 3, 3-trisulfonic acid trisodium salt, tris (2, 5-dimethylphenyl) phosphine, tri-p-tolylphosphine, 4-diphenylphosphinobenzoic acid, 2-diphenylphosphinobenzoic acid, diphenyl (o-methoxyphenyl) phosphine, 4-diphenylphosphinobenzenesulfonic acid sodium salt, 4- (dimethylamino) triphenylphosphine, 3-diphenylphosphinophenyl benzene, 2-diphenylphosphinobenzonitrile, benzene, Bis (4-methoxyphenyl) phenylphosphine, bis (p-sulfonylphenyl) phenylphosphine dipotassium salt dihydrate, bis (3-sulfophenyl) (3, 5-di-trifluoromethylphenyl) phosphonic acid disodium monohydrate, bis (4-trifluoromethylphenyl) (3-sulfophenyl) phosphine, sodium 3-diphenylphosphinobenzenesulfonate dihydrate, 1-bis (diphenylphosphino) methane, 1, 2-bis (dimethylphosphino) ethane, 1, 2-bis (diphenylphosphino) ethane, 1, 3-bis (diphenylphosphino) propane, 1, 4-bis (diphenylphosphino) butane, 2, 3-bis (diphenylphosphino) butane, diphenylcyclohexylphosphine, diphenylethoxyphosphine, methyldiphenylphosphine, ethyldiphenylphosphine, carboxymethyldiphenylphosphine, phenyldicyclohexylphosphine, sodium bis (3-sulfophenyl) phosphonate monohydrate, disodium salt of bis (3-sulfophenyl) (3-trifluoromethylphenyl) phosphine, disodium salt of bis (4-trifluoromethylphenyl) phosphine, 1, 2-bis (diphenylphosphine) ethane, 2-bis (dimethylphosphine, bis (diphenylphosphine) ethane, bis (diphenylcyclohexylphosphine, bis (diphenylethyphosphine), bis (diphenylphosphine, bis (diphenylethyphosphine), bis (diphenylethyiene, phenyl) propane, bis (ethoxyphosphine, bis (ethyldiphenylphosphine, phenyl) propane, bis (ethoxyphosphine, bis (carboxymethyldiphenylphosphine, bis (phenyldicyclohexylphosphine), bis (ethoxyphosphine), bis (ethoxyphosphine, bis (ethylbis (ethoxyphosphine), bis (carboxymethyldiphenylphosphine), bis (ethylbis (p-bis (phenyldicyclohexylphosphine), bis (ethylbis (p-bis (p-bis (p-bis (p-ethyl) phosphine), bis (p-, Dimethylphenylphosphine, di-tert-butylphosphine, 2- (di-tert-butylphosphine) biphenyl, 1 '-binaphthyl-2, 2' -bisdiphenylphosphine, 2-dicyclohexylphosphine-2, 4, 6-triisopropylbiphenyl, 2-dicyclohexylphosphine-2 '-methylbiphenyl, 2-dicyclohexylphosphine-2', 6 '-diisopropoxy-1, 1' -biphenyl, 2-dicyclohexylphosphino-2 ',6' -dimethoxybiphenyl, 2-di-tert-butylphosphino-2 ',4',6 '-triisopropylbiphenyl, 2-di-tert-butylphosphino-2' -methylbiphenyl, 2-di-tert-butylphosphino-2 '- (N, N-dimethylamino) biphenyl, N-tert-butylphosphine, N-propylbiphenyl, N-tert-propylbiphenyl, N-tert-butylphosphine-2' -methylbiphenyl, N-butylbiphenyl, N-butylphosphino-2-isopropylbiphenyl, 2-dicyclohexylphosphino-o-2 '- (N, N-dimethylamino) biphenyl, 2- (di-tert-butylphosphino) biphenyl, 2-diphenylphosphino-2' - (N, N-dimethylamino) biphenyl, 2-dicyclohexylphosphine-2, 6-diisopropyl-3-sulfo-1, 1-biphenyl sodium salt hydrate, 4- (2, 6-dimethoxyphenyl) -3- (1, 1-dimethylethyl) -2, 3-dihydro-1, 3-benzoxaphosphocycloheptatriene, 2- (dicyclohexylphosphino) biphenyl, [4- (N, N-dimethylamino) phenyl ] di-tert-butylphosphine, tris (2-carboxyethyl) phosphine, tris (tert-butylamino) phosphine, bis (tert-butylamino) phosphine), bis (2-carboxyethyl) phosphine, bis (tert-butylamino) phosphonium), bis (tert-butylamino) phosphonium), bis (2-butylamino) phosphonium), bis (tert-propylphosphonium), bis (tert-butylamino) phosphonium or a salt, or a salt of a compound of a (I, or a salt of a compound of a, Tris (3-hydroxypropyl) phosphine, tricyclohexylphosphine, 4, 5-bis (diphenylphosphino) -9, 9-dimethylxanthene or one or a mixture of more of 1,1' -bis (di-tert-butylphosphino) ferrocene; when a Pd (0) catalyst is used, the ligand may not be used.
The alkali is one or a mixture of more of sodium borate, potassium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium acetate, sodium fluoride, potassium fluoride, cesium fluoride, lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, cesium carbonate, sodium bicarbonate, potassium bicarbonate, pyrrolidine, diisopropylethylamine, triethylamine, diisopropylamine, diethylamine, pyridine, piperidine, tetrabutylammonium fluoride, tetrabutylammonium chloride, tetrabutylammonium bromide and tetrabutylammonium iodide; preferably, the base is diisopropylamine.
The On-DNA terminal alkyne compound of the invention can be prepared from DNA-NH2And a binary compound containing carboxylic acid terminal alkyne through in-situ condensation reaction.
The invention provides a new method for the application of On-DNA Sonogashira coupling reaction in the construction of a DNA coding compound library, and the invention can increase the diversity of the DNA coding compound library by using low-cost and easily-obtained small-molecule aryl halide with good stability and multiple reagent types as a reaction reagent, particularly only CuAAC is used for mature capping reaction before an On-DNA terminal alkyne compound, and the method is suitable for the production of DNA coding compound libraries with large-batch porous plates.
Drawings
FIG. 1 shows a DNA-NH sequence of the present invention2To prepare a chemical reaction formula of the On-DNA alkyl or aryl terminal alkyne compound.
FIG. 2 shows the chemical reaction formula and conversion rate distribution (conversion rate is based On the TIC peak area On LCMS-LTQ) of the On-DNA aryl alkyne compound prepared from the On-DNA aryl terminal alkyne compound, wherein the ordinate is the number of reagents, the abscissa is the conversion rate interval, and if 0-30%, the conversion rate is more than 0% and less than or equal to 30%.
FIG. 3 is a representative structural formula and reaction yield of small-molecule aryl halide of an On-DNA aryl alkyne compound prepared from an On-DNA aryl terminal alkyne compound.
FIG. 4 shows the chemical reaction formula and conversion rate distribution (conversion rate is based On the TIC peak area On LCMS-LTQ) of On-DNA aryl alkyne compound prepared from On-DNA alkyl terminal alkyne compound of the present invention, wherein the ordinate is the number of reagents, the abscissa is the conversion rate interval, and if 0-30%, the conversion rate is more than 0% and less than or equal to 30%.
FIG. 5 is a representative structural formula and reaction yield of small molecule aryl halide of On-DNA aryl alkyne compound prepared from On-DNA alkyl terminal alkyne compound.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Example 1 Synthesis of On-DNA alkyl/aryl terminal alkynes
DNA-NH2(for example, the initial headpiece mentioned in patent CN 108070009A) was dissolved in 250mM boric acid buffer at pH 9.5 to prepare a 1mM solution, which was reacted with 3-ethynyl-benzoic acid or 10-undecyynoic acid using EDCI as a condensing agent and an s-NHS condensation activator to give the corresponding On-DNA terminal alkyne compound (reference: nat. chem.,2015,7,3,241), conversion>After 90% ethanol precipitation treatment, the mixture was ultrafiltered using a 3K Millpore ultrafilter tube, concentrated and dried before being used directly in the next reaction (see FIG. 1).
Example 2 Synthesis of On-DNA Arylacetylene Compound
DNA-Ar-C.ident.C was dissolved in ultrapure water to prepare a 1mM concentration solution, which was dispensed into 96-well plates (10. mu.L, 10nmol,1mM aqueous solution), an aryl bromide (5.0. mu.L, 5000nmol,1000mM dimethylacetamide solution, 500eq.) was added, the solution was centrifuged to settle, vortexed, centrifuged again, then nitrogen was replaced 3 times, diisopropylamine (DIPA, 4.0. mu.L, 28.3. mu. mol,2830eq., d. 0.717g/mL) and tetratriphenylphosphine palladium (Pd (PPh)3)44.0 μ L of 10mM dimethylacetamide solution, 4eq.), centrifuged to settle the solution, vortexed and mixed, centrifuged again, replaced with nitrogen again 3 times, sealed, and reacted in a 96-well plate in a PCR instrument at 65 ℃ for 3 hours (cover temperature: 85 deg.C).
Removing palladium: after completion of the reaction, a sodium diethyldithiocarbamate solution (12.0. mu.L, 1200nmol,100mM aqueous solution, 120eq.) was added to each well of a 96-well plate, centrifuged to let the solution settle, vortexed and mixed, centrifuged again, and then the 96-well plate was reacted in a PCR instrument at 80 ℃ for 30 minutes (cover temperature: 105 ℃ C.), after completion of the reaction, a precipitate was generated, centrifuged at 4000G for 5 minutes, and the supernatant was transferred to a new 96-well plate to be subjected to ethanol precipitation.
Ethanol precipitation: adding 5M sodium chloride solution with the volume of 10% of that of the reaction solution into each hole of a 96-hole plate, sealing a membrane, oscillating and uniformly mixing, adding cold absolute ethyl alcohol with the volume of 3 times of that of the reaction solution and stored at the temperature of-20 ℃, freezing the mixture in a refrigerator at the temperature of-80 ℃ for 2 hours, taking out the mixture, centrifuging the mixture at the temperature of 4 ℃ for 30 minutes by using a centrifugal force of 4000G, absorbing and removing supernatant, dissolving precipitates in deionized water, and then carrying out vacuum freeze-drying at the temperature of-40 ℃ to obtain a product, detecting OD (optical density) by using an enzyme labeling instrument to confirm the recovery rate, and simultaneously detecting LC-MS (liquid chromatography-mass spectrometry) to confirm the conversion rate of each micromolecule.
1605 aryl bromide reagents reacted with the On-DNA aryl terminal alkyne compound are tested in total, the reagent with the conversion rate of more than 50 percent accounts for more than 55 percent, the reagent with the conversion rate of more than 70 percent accounts for more than 27 percent (see figure 2), wherein the conversion rate of the aryl bromide binary or multi-component compound containing carboxyl, aldehyde or ketone functional groups is better, the obtained DNA aryl alkyne compound library can continue to use the remaining functional groups to carry out capping reaction to obtain different sub-libraries so as to increase the diversity of the DNA coding compound library, and the representative structural formula and the yield of part of the aryl bromide reagents are shown in figure 3.
Example 3 Synthesis of On-DNA Alkylalkyne Compounds
DNA-(CH2)8Dissolving C.ident.C in ultrapure water to give a 1mM concentration solution, dispensing into 96-well plates (10. mu.L, 10nmol,1mM aqueous solution), and adding aryl bromide (5.0)μL,5000nmol,1000mM dimethylacetamide solution, 500eq.), centrifuging to let the solution settle, mixing by vortexing, centrifuging again, replacing nitrogenQiqi for 3 times, and then DIPA (4.0 μ L, 28.3)μmol,2830eq, d 0.717g/mL) and Pd (PPh)3)4(4.0μL,10mM dimethylacetamide solution, 4eq.), centrifuging to settle the solution, vortexing and mixing, centrifuging again, replacing nitrogen again for 3 times, sealing the membrane, reacting 96-well plate in a PCR instrument at 65 ℃ for 3 hours (cover temperature: 85 deg.C).
Removing palladium: after completion of the reaction, a sodium diethyldithiocarbamate solution (12.0) was added to each well of a 96-well plateμL,1200nmol,100mM aqueous solution, 120eq.), centrifugation, allowing the solution to settle, vortexing and mixing, re-centrifugation, and reaction of 96-well plates in a PCR instrument at 80 ℃ for 30 minutes (cover temperature: at 105 ℃ C.), a precipitate was generated, centrifuged at 4000G for 5 minutes, and the supernatant was transferred to a new 96-well plate for ethanol precipitation.
Ethanol precipitation: adding 5M sodium chloride solution with the volume of 10% of that of the reaction solution into each hole of a 96-hole plate, sealing a membrane, oscillating and uniformly mixing, adding cold absolute ethyl alcohol with the volume of 3 times of that of the reaction solution and stored at the temperature of-20 ℃, freezing the mixture in a refrigerator at the temperature of-80 ℃ for 2 hours, taking out the mixture, centrifuging the mixture at the temperature of 4 ℃ for 30 minutes by using a centrifugal force of 4000G, absorbing and removing supernatant, dissolving precipitates in deionized water, and then carrying out vacuum freeze-drying at the temperature of-40 ℃ to obtain a product, detecting OD (optical density) by using an enzyme labeling instrument to confirm the recovery rate, and simultaneously detecting LC-MS (liquid chromatography-mass spectrometry) to confirm the conversion rate of each micromolecule.
A total of 1605 aryl bromide reagents reacted with the On-DNA alkyl terminal alkyne compound were also tested, 35% reagents with conversion > 50% (see FIG. 4), wherein the conversion of the aryl bromide binary or multi-element compound containing aldehyde or ketone functional group is better, and the representative structural formula and yield of part of the aryl bromide reagents are shown in FIG. 5.
In summary, the above embodiments and drawings are only preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (16)
1. A method for preparing an On-DNA aryl alkyne compound in the construction of a DNA coding compound library is characterized in that 1 molar equivalent of an On-DNA terminal alkyne compound aqueous solution with the concentration of 0.1-5.0 mM is added with 1-100 molar equivalents of a palladium catalyst, 0-100 molar equivalents of a ligand, 10-5000 molar equivalents of alkali and 10-2000 molar equivalents of a small molecular aryl halide reagent, and the reaction is carried out at 0-100 ℃ for 0.5-24 hours until the reaction is finished;
wherein the On-DNA terminal alkyne compound has a structural formula
The structural formula of the prepared On-DNA aryl alkyne compound is shown as 
(ii) a The DNA in the structural formula is a single-stranded or double-stranded nucleotide chain obtained by polymerizing artificially modified and/or unmodified nucleotide monomers, the Ar group is a residual part of a small-molecular aryl halide reagent with the molecular weight of below 1000, which can participate in the reaction, on a product, and the small-molecular aryl halide is aryl iodide or aryl bromide.
2. The method of claim 1, wherein the molar concentration of the On-DNA terminal alkyne compound is 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, or 5.0 mM.
3. The method of claim 1, wherein the On-DNA terminal alkyne compound is dissolved in an aqueous mixed solvent of any one or more of acetonitrile, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide, methanol, ethanol, t-butanol, isopropanol, tetrahydrofuran, 1, 4-dioxane, an inorganic salt buffer, an organic acid buffer, and an organic base buffer, and the water content in the final reaction solution is not less than 20%.
4. The process of claim 1, wherein the palladium catalyst is palladium chloride, palladium bromide, palladium iodide, bis (acetonitrile) palladium dichloride, ethylenediamine palladium chloride, palladium trifluoroacetate, palladium acetate, palladium sulfate, palladium nitrate, palladium dipropionate, palladium acetylacetonate, palladium hexafluoroacetylacetonate, dichloro (norbornadiene) palladium, (1, 5-cyclooctadiene) palladium dichloride, dichloro (N, N, N, N-tetramethylethylenediamine) palladium, dichloro (1, 10-phenanthroline) palladium, bis (dibenzylideneacetone) palladium, tris (dibenzylideneacetone) dipalladium, tetrakis (acetonitrile) bis (trifluoromethanesulfonic) palladium, tetrakis (triphenylphosphine) palladium, tetrakis (tri-o-tolylphosphine) palladium, triphenylphosphine palladium acetate, bis (tri-o-tolylphosphine) palladium, bis [1, 2-bis (diphenylphosphino) ethane ] palladium, palladium chloride, palladium (trifluoroacetate), palladium, bis (norbornadiene) dichloride, dichloro (1, 5-cyclooctadiene) palladium, dichloro (N, N, N-tetramethylethylenediamine palladium (tetramethylethylenediamine) palladium, dichloro (1, N, N-tetramethylethylenediamine) palladium, N-tetramethylethylenediamine palladium, bis (triphenylphosphine) palladium, palladium (triphenylphosphine) acetate, palladium, and palladium (tris-tolylphosphine, palladium (or a mixture of, One or a mixture of more of bis (tri-tert-butylphosphine) palladium, dihydrodichlorobis (di-tert-butylhydroxyphosphinylidene) palladium, bis (triphenylphosphine) palladium dichloride, (1, 3-bis (diphenylphosphino) propane) palladium chloride, [1,1 '-bis (diphenylphosphino) ferrocene ] palladium dichloride dichloromethane complex, dibromobis (tri-tert-butylphosphino) dipalladium, 1' -bis (di-tert-butylphosphino) ferrocene palladium dichloride and bis (tricyclohexylphosphine) palladium dichloride.
5. The method of claim 1, wherein the molar equivalent of the palladium catalyst is 1 to 100 equivalents.
6. The method of claim 1, wherein the molar equivalent of the palladium catalyst is 1 equivalent, 2 equivalents, 3 equivalents, 4 equivalents, 5 equivalents, 6 equivalents, 7 equivalents, 8 equivalents, 9 equivalents, 10 equivalents, 20 equivalents, 30 equivalents, 40 equivalents, 50 equivalents, 60 equivalents, 70 equivalents, 80 equivalents, 90 equivalents, or 100 equivalents.
7. The method of claim 1, wherein the ligand is triphenylphosphine, tris [3, 4-bis (trifluoromethyl) phenyl ] phosphine, tris (4-fluorophenyl) phosphine, tris (3-bromophenyl) phosphine, tris (2-methoxyphenyl) phosphine, tris (4-methoxy-3, 5-xylyl) phosphine, tris (4-dimethylaminophenyl) phosphine, tris (4-chlorophenyl) phosphine, tris (3-chlorophenyl) phosphine, triphenylphosphine-3, 3, 3-trisulfonic acid trisodium salt, tris (2, 5-xylyl) phosphine, tri-p-tolylphosphine, 4-diphenylphosphinobenzoic acid, 2-diphenylphosphinobenzoic acid, diphenyl (o-methoxyphenyl) phosphine, sodium 4-diphenylphosphinobenzenesulfonate, 4- (dimethylamino) triphenylphosphine, 3-diphenylphosphinophenyl, 2-diphenylphosphinophenyl, bis (4-methoxyphenyl) phenylphosphine, bis (p-sulfonylphenyl) phenylphosphine dipotassium salt dihydrate, bis (3-sulfophenyl) (3, 5-bis-trifluoromethylphenyl) phosphonic acid disodium monohydrate, bis (4-trifluoromethylphenyl) (3-sulfophenyl) phosphine, sodium 3-diphenylphosphinobenzenesulfonate dihydrate, 1-bis (diphenylphosphino) methane, 1, 2-bis (dimethylphosphino) ethane, 1, 2-bis (diphenylphosphino) ethane, 1, 3-bis (diphenylphosphino) propane, 1, 4-bis (diphenylphosphino) butane, 2, 3-bis (diphenylphosphino) butane, diphenylcyclohexylphosphine, diphenylethoxyphosphine, methyldiphenylphosphine, Ethyldiphenylphosphine, carboxymethyldiphenylphosphine, phenyldicyclohexylphosphine, dimethylphenylphosphine, di-tert-butylphenyl-phosphine, 2- (di-tert-butylphosphino) biphenyl, 1' -binaphthyl-2, 2' -bisdiphenylphosphine, 2-dicyclohexylphosphine-2, 4, 6-triisopropylbiphenyl, 2-dicyclohexylphosphine-2 ' -methylbiphenyl, 2-dicyclohexylphosphine-2 ',6' -diisopropoxy-1, 1' -biphenyl, 2-dicyclohexylphosphine-o-2 ',6' -dimethoxybiphenyl, 2-di-tert-butylphosphino-2 ',4',6' -triisopropylbiphenyl, 2-di-tert-butylphosphino-2 ' -methylbiphenyl, 2-di-tert-butylphosphino-2 ' - (N), n-dimethylamino-biphenyl, 2-dicyclohexylphosphino-o-2 '- (N, N-dimethylamino) biphenyl, 2- (di-tert-butylphosphino) biphenyl, 2-diphenylphosphino-2' - (N, N-dimethylamino) biphenyl, 2-dicyclohexylphosphine-2, 6-diisopropyl-3-sulfo-1, 1-biphenyl sodium salt hydrate, 4- (2, 6-dimethoxyphenyl) -3- (1, 1-dimethylethyl) -2, 3-dihydro-1, 3-benzoxaphosphepin, 2- (dicyclohexylphosphino) biphenyl, [4- (N, N-dimethylamino) phenyl ] di-tert-butylphosphine, N-cyclohexylphosphino-2-phenyl-1, 3-dihydrophosphepin, N-dimethylphosphino-1, 3-dihydrophosphepin, N-dimethylphosphino-2, N-di-tert-butylphosphine, N-di-ethylphosphine, N-4-1-dimethylphosphino-2, N-2-1, 6-di-isopropyl-3-sulfonic acid, 1-diphenyl, 4-p, Tris (2-carboxyethyl) phosphine, tris (3-hydroxypropyl) phosphine, tricyclohexylphosphine, 4, 5-bisdiphenylphosphine-9, 9-dimethylxanthene or one or more mixtures of 1,1' -bis (di-tert-butylphosphine) ferrocene.
8. The method of claim 1, wherein the ligand is not used when the palladium catalyst is used.
9. The method of claim 1, wherein the base is one or more of sodium borate, potassium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium acetate, sodium fluoride, potassium fluoride, cesium fluoride, lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, cesium carbonate, sodium bicarbonate, potassium bicarbonate, pyrrolidine, diisopropylethylamine, triethylamine, diisopropylamine, diethylamine, pyridine, piperidine, tetrabutylammonium fluoride, tetrabutylammonium chloride, tetrabutylammonium bromide, tetrabutylammonium iodide.
10. The method of claim 1, wherein the equivalent of the base is 10 equivalents, 20 equivalents, 30 equivalents, 40 equivalents, 50 equivalents, 60 equivalents, 70 equivalents, 80 equivalents, 90 equivalents, 100 equivalents, 200 equivalents, 300 equivalents, 400 equivalents, 500 equivalents, 600 equivalents, 700 equivalents, 800 equivalents, 900 equivalents, 1000 equivalents, 1500 equivalents, 2000 equivalents, 2500 equivalents, 3000 equivalents, 3500 equivalents, 4000 equivalents, 4500 equivalents, or 5000 equivalents.
11. The process of claim 1, wherein the equivalents of the aryl halide reagent are 10 equivalents, 20 equivalents, 30 equivalents, 40 equivalents, 50 equivalents, 60 equivalents, 70 equivalents, 80 equivalents, 90 equivalents, 100 equivalents, 150 equivalents, 200 equivalents, 250 equivalents, 300 equivalents, 350 equivalents, 400 equivalents, 450 equivalents, 500 equivalents, 550 equivalents, 600 equivalents, 650 equivalents, 700 equivalents, 750 equivalents, 800 equivalents, 850 equivalents, 900 equivalents, 950 equivalents, 1000 equivalents, 1500 equivalents, or 2000 equivalents.
12. The method of claim 1, wherein the reaction temperature of the reaction is 20 to 100 ℃.
13. The process of claim 1, wherein the reaction temperature of the reaction is 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃, 70 ℃, 75 ℃, 80 ℃, 85 ℃, 90 ℃, 95 ℃, or 100 ℃.
14. The method of claim 1, wherein the reaction is for a reaction time of 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours.
15. The method of claim 1, wherein the method is used in a batch multiwell plate operation.
16. The method of claim 1, wherein the method is used for the synthesis of a library of DNA-encoding compounds for multi-well plates.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910609568.6A CN110331447B (en) | 2019-07-08 | 2019-07-08 | Method for preparing On-DNA aryl alkyne compound in construction of DNA coding compound library |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910609568.6A CN110331447B (en) | 2019-07-08 | 2019-07-08 | Method for preparing On-DNA aryl alkyne compound in construction of DNA coding compound library |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110331447A CN110331447A (en) | 2019-10-15 |
| CN110331447B true CN110331447B (en) | 2022-05-27 |
Family
ID=68144375
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910609568.6A Active CN110331447B (en) | 2019-07-08 | 2019-07-08 | Method for preparing On-DNA aryl alkyne compound in construction of DNA coding compound library |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110331447B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110759958B (en) * | 2019-10-30 | 2023-03-14 | 上海药明康德新药开发有限公司 | Method for synthesizing On-DNA phosphoramidate compound in construction of DNA coding compound library |
| CN111620921B (en) * | 2020-05-25 | 2023-06-13 | 上海药明康德新药开发有限公司 | Method for preparing On-DNA amide compound by oxidative amidation in construction of DNA coding compound library |
| CN114075257B (en) * | 2020-08-11 | 2023-08-22 | 成都先导药物开发股份有限公司 | Method for preparing arylamine compound from On-DNA aryl halide |
| CN112851733A (en) * | 2021-01-15 | 2021-05-28 | 重庆大学 | Method for preparing C-C coupling product based On-DNA aryl diazonium salt intermediate |
| CN114106072B (en) * | 2021-12-13 | 2023-11-14 | 康龙化成(宁波)科技发展有限公司 | Method for synthesizing DNA-iodoalkyne from DNA-terminal alkyne |
| CN114195842B (en) * | 2021-12-17 | 2024-07-19 | 康龙化成(宁波)科技发展有限公司 | Terminal alkynyl deoxyribonucleic acid and self-coupling method thereof |
| CN114957366B (en) * | 2021-12-17 | 2023-11-10 | 康龙化成(宁波)科技发展有限公司 | 1, 3-diacetylene lead compound and synthesis method thereof |
| CN114292304B (en) * | 2022-01-12 | 2024-07-09 | 康龙化成(宁波)科技发展有限公司 | Method for synthesizing aryl alkyne lead compound |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101544892A (en) * | 2009-05-07 | 2009-09-30 | 石家庄永生华清液晶有限公司 | Method for synthesizing diaryl acetylene monomer liquid crystal |
| CN109439710A (en) * | 2018-12-07 | 2019-03-08 | 上海药明康德新药开发有限公司 | T4 DNA ligase buffer and its application for enzyme chain reaction in the synthesis of DNA encoding compound library |
| WO2019060857A1 (en) * | 2017-09-25 | 2019-03-28 | Plexium, Inc. | Oligonucleotide encoded chemical libraries |
-
2019
- 2019-07-08 CN CN201910609568.6A patent/CN110331447B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101544892A (en) * | 2009-05-07 | 2009-09-30 | 石家庄永生华清液晶有限公司 | Method for synthesizing diaryl acetylene monomer liquid crystal |
| WO2019060857A1 (en) * | 2017-09-25 | 2019-03-28 | Plexium, Inc. | Oligonucleotide encoded chemical libraries |
| CN109439710A (en) * | 2018-12-07 | 2019-03-08 | 上海药明康德新药开发有限公司 | T4 DNA ligase buffer and its application for enzyme chain reaction in the synthesis of DNA encoding compound library |
Non-Patent Citations (2)
| Title |
|---|
| DNA编码化合物库在药物筛选和发现中的研究与应用;徐力昆等;《国际药学研究杂志》;20181031;第45卷(第10期);第736-742页 * |
| Palladium-Promoted DNA-Compatible Heck Reaction;Xuan Wang et al.;《Organic Letters》;20190124;第21卷;第719-723页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110331447A (en) | 2019-10-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110331447B (en) | Method for preparing On-DNA aryl alkyne compound in construction of DNA coding compound library | |
| CN110359097B (en) | Method for obtaining On-DNA aromatic hydrocarbon compound through Suzuki coupling reaction in construction of DNA coding compound library | |
| CN110041390B (en) | Method for synthesizing On-DNA 1,2, 3-triazole compound in DNA coding compound library | |
| CN110105408B (en) | Synthetic method of On-DNA aryl azide in construction of DNA coding compound library | |
| Ruff et al. | An amphiphilic polymer-supported strategy enables chemical transformations under anhydrous conditions for DNA-encoded library synthesis | |
| CN109680342A (en) | The method that On-DNA virtue nitro compound is reduced into On-DNA aromatic amine compound in DNA encoding compound library | |
| Lee et al. | Polymeric chiral phase-transfer catalysts derived from cinchona alkaloids for enantioselective synthesis of α-amino acids | |
| CN110818749A (en) | Method for synthesizing On-DNA aryl sulfonamide compounds in construction of DNA coding compound library | |
| EP1299722A1 (en) | Methods for encoding combinatorial libraries | |
| Merrington et al. | Supported diazonium salts—convenient reagents for the combinatorial synthesis of azo dye | |
| CN110820049A (en) | Method for synthesizing On-DNA aryl iodide in construction of DNA coding compound library | |
| CN110066308A (en) | Synthetic method for the On-DNA sulfamide compound in the building of DNA encoding compound library | |
| Lu et al. | Photoredox catalysis in alkene and alkyne alkylsulfonylations: the construction of Markovnikov selective α-sulfones | |
| CN110079867B (en) | On-DNA wittig reaction method in DNA coding compound library and prepared On-DNA olefin compound | |
| CN113004361B (en) | Method for synthesizing On-DNA pyrazole compound | |
| CN112851733A (en) | Method for preparing C-C coupling product based On-DNA aryl diazonium salt intermediate | |
| Patterson et al. | Triarylmethyl Cation‐Catalyzed Three‐Component Coupling for the Synthesis of Unsymmetrical Bisindolylmethanes | |
| Shi et al. | A combinatorial synthesis of tyrphostins via the “directed sorting” method | |
| CN111909232A (en) | Method for preparing On-DNA1, 2-amino alcohol compound through visible light catalysis in construction of DNA coding compound library | |
| CN113388894A (en) | Method for functionalizing aryl olefin of On-DNA in construction of DNA coding compound library | |
| CN112661803A (en) | Synthetic method of On-DNA 4-amino quinazoline compound in construction of DNA coding compound library | |
| CN112851732B (en) | Method for synthesizing On-DNA 2-carboxyl-3-amino arylthiophene compound | |
| EP0817771A1 (en) | Photolytically cleavable encoding and linking agents for use in combinatorial chemistry | |
| CN112831843A (en) | One-pot On-DNA Suzuki reaction method | |
| CN112920246B (en) | Method for synthesizing On-DNA1,4-thiazepine compound |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |