阅读说明：本技术 DNA编码化合物库构建中On-DNA氨基磷酸酯化合物的合成方法 (Method for synthesizing On-DNA phosphoramidate compound in construction of DNA coding compound library ) 是由 刘炜 邓伟 孙赛赛 喻春燕 龚平秀 陈琳琳 范梅 吴阿亮 李科 蒯乐天 杨洪芳 于 2019-10-30 设计创作，主要内容包括：本发明公开了一种DNA编码化合物库中On-DNA氨基磷酸酯化合物的制备方法,将On-DNA叠氮化合物与亚磷酸三酯混合在一定温度下反应一段时间后得到DNA氨基磷酸酯化合物。本发明反应条件温和、成本低、操作方便、收率高,适合于多孔板进行的DNA编码化合物库的合成,可以让DNA编码胺基化合物库通过该方法盖帽得到DNA编码氨基磷酸酯类化合物库,提供了一种增加DNA编码氨基化合物库的多样性的方法。(The invention discloses a preparation method of an On-DNA phosphoramidate compound in a DNA coding compound library, which comprises the steps of mixing an On-DNA azide compound and phosphotriester, reacting for a period of time at a certain temperature to obtain the DNA phosphoramidate compound. The method has the advantages of mild reaction conditions, low cost, convenient operation and high yield, is suitable for synthesizing the DNA coding compound library by using a porous plate, can obtain the DNA coding phosphoramidate compound library by using the DNA coding amino compound library through the method, and provides a method for increasing the diversity of the DNA coding amino compound library.)

1. A method for constructing an On-DNA phosphoramidate compound in a DNA coding compound library is characterized in that an On-DNA azide compound with the molar concentration of 0.1-2.0 mM and a phosphite triester solution with the molar equivalent of 10-500 are mixed and reacted at the temperature of 20-100 ℃ for 1-24 hours until the reaction is finished;

wherein the structural formula of the On-DNA azide compound is as follows: DNA-N₃The structural formula of the On-DNA phosphoramidate compound is as follows: DNA-NH-P ═ O (OR)₂；

Wherein R is halogen, amido, C₁-C₈Alkyl radical, C₃-C₈Cycloalkyl radical, C₁-C₈Alkylene radical, C₁-C₈Alkynyl radical, C₁-C₈Alkoxy radical, C₃-C₈Any one to many random combinations of alkyl silicon base and phenyl;

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.

2. The method of claim 1, wherein the On-DNA azide is dissolved in an aqueous solution at a molar concentration of 0.1 to 2.0 mM; preferably, the molar concentration of the On-DNA azide aqueous solution is 1.0 mM.

3. The method according to claim 1, wherein the solution is an aqueous mixed solvent containing any one or more of acetonitrile, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethylsulfoxide, methanol, ethanol, t-butanol, isopropanol, tetrahydrofuran, an inorganic salt buffer, an organic acid buffer, and an organic base buffer; preferably, the on-DNA azide is soluble in water.

4. The method of claim 1, wherein the molar equivalents of the phosphite triester are 10 to 500 equivalents; preferably, the molar equivalent of the phosphite triester is 200 equivalents.

5. The method of claim 1, wherein the reaction temperature of the reaction is 20 to 100 ℃; preferably, the reaction temperature of the reaction is 40 ℃.

6. The method of claim 1, wherein the reaction time is 1 to 24 hours; preferably, the reaction time of the reaction is 16 hours.

7. The method of claim 1, wherein the method is used for a batch multi-well plate operation; preferably, the method is used for the synthesis of libraries of DNA-encoding compounds for multiwell plates.

Technical Field

The invention belongs to the technical field of DNA coding compound libraries, and particularly relates to a method for preparing an On-DNA phosphoramidate compound from an On-DNA azide in a DNA coding compound library.

Background

The concept of libraries of DNA-Encoded Library (DEL) was proposed in 1992 by the teaching of Sydney Brenner and Richard Lerner of the Scripps institute of America (reference: Proc. Natl.Acad.Sci.,1992,89,5381) by linking an organic small molecule reagent to a unique sequence of DNA at the molecular level, rapidly constructing a large Library of compounds each consisting of different organic small molecule reagent residues and identified by DNA of a corresponding unique base sequence using a combinatorial chemistry "combinatorial-resolution" strategy over two to many cycles, affinity-screening a small Library of DNA-Encoded compounds to a target, washing away Library molecules that are not adsorbed to the target, leaving Library molecules adsorbed to the target and then washing away, where the resulting Library molecule concentrations are low and difficult to analyze and identify by conventional means, polymerase chain Reaction (polymerase chain Reaction, PCR for short) can be carried out to copy and amplify the DNA part in the obtained library molecule adsorbed with the target until the obtained DNA quantity can be identified by a DNA sequencer, the sequenced data is decoded by a relation table between small molecule reagents and DNA base sequences established when a DNA coding compound library is constructed, then the small molecule reagents corresponding to specific compounds corresponding to potential active molecules are found, then the small molecule reagents are combined together by a traditional organic synthesis method to obtain the screened target molecules, and the biological activity of the target molecules on the target 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 leaders, 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 massive compounds by using a combinatorial chemistry method.

Besides the DNA starting fragment (see the invention patents of the present company: CN108070009A, CN109868268A), a large number of DNA tags and small organic molecule reagents which can be reacted in a certain order are required. The DNA tag code can be obtained by a computer program (see the present invention: CN107958139A), and a primer of a specific DNA base sequence 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 108959855A).

One of the most important works in the field of DEL libraries 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 aqueous phase, pH, temperature, metal ion concentration and inorganic salt concentration, on-DNA chemical reactions with less DNA damage, better recovery rate and wide substrate adaptability are required for large-scale application in the synthesis of DNA coding compound libraries. The number of the on-DNA chemical reactions reported in the prior art is about 60, and each reaction condition is one or more than ten, so that under the same other conditions, the more the 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 DNA coding compound library is, and the more the diversity of the obtained DNA coding compound library is.

TABLE 1 On-DNA chemistry reaction types and specific conditions that can be used for DRCL construction

In the On-DNA chemical reaction, the selection of buffer is very important, and we have determined the preparation and quality inspection methods of several common buffers (see the invention patent of the company: CN109456368A for details) and provided several specific On-DNA chemical reactions under the condition of the buffer (see the invention patent of the company: CN109680342A for details). The most commonly used bonding chemistry 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).

Due to the limitations of the above-described on-DNA chemistry, it is currently not possible to directly obtain a wide variety of DNA-encoding compound molecules by synthesis of libraries of DNA-encoding compounds. In order to introduce the synthesis of small molecule phosphoramidates, which is currently the most popular study in the field of pharmaceutical chemistry, into the synthesis of our library of DNA-encoding compounds, we studied and developed the method of the present invention.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a synthetic method for obtaining an On-DNA phosphoramidate compound by one-step reaction of an On-DNA azide in the construction of a DNA coding compound library, which has the advantages of mild reaction conditions, high selectivity, high yield and simple post-treatment, is suitable for the production of the DNA coding compound library, and can obviously improve the molecular diversity of the compound library.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a synthetic method of an On-DNA phosphoramidate compound in the construction of a DNA coding compound library is characterized in that the On-DNA azide compound DNA-N with the molar concentration of 0.1-2.0 mM is used₃Mixing with 10-500 molar equivalent of phosphite triester solution, and reacting at 20-100 ℃ for 1-24 hours until the reaction is finished, wherein the reaction structural formula is as follows:

wherein the structural formula of the On-DNA azide compound is as follows: DNA-N₃The structural formula of the On-DNA phosphoramidate compound is as follows: DNA-NH-P ═ O (OR)₂；

Wherein R is halogen, amido, C₁-C₈Alkyl radical, C₃-C₈Cycloalkyl radical, C₁-C₈Alkylene radical, C₁-C₈Alkynyl radical, C₁-C₈Alkoxy radical, C₃-C₈Any one to many random combinations of alkyl silicon base and phenyl;

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;

wherein the solvent is a water-containing mixed solvent containing any one or more of acetonitrile, dimethylformamide, dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, methanol, ethanol, tert-butyl alcohol, isopropanol, tetrahydrofuran, an inorganic salt buffer solution, an organic acid buffer solution and an organic base buffer solution; preferably, the on-DNA azide is soluble in water.

The invention provides a method for directly synthesizing DNA coding phosphoramidate in a DNA coding compound library, which aims to expand the diversity of the DNA coding compound library of the company and enable the diversity of the library molecule synthesis method of the DNA coding compound library to cover the main hot spot field of the current research field of small molecule drugs, so that the library molecules of the obtained DNA coding compound library can better meet the market demands.

The method has the advantages of mild reaction conditions, high selectivity, high yield and simple post-treatment, is suitable for the production of large-scale multi-plate DNA coding compound libraries, and can obviously improve the diversity of the molecules of the current DNA coding compound libraries.

Drawings

FIG. 1 shows a method for preparing a raw material, DNA-NH, for the method of the present invention₂The compound 1 and a binary compound containing aromatic nitrocarboxylic acid are condensed by EDCI as a condensing agent and s-NHS as a condensation activating agent to generate an on-DNA aromatic nitro compound 2, and the on-DNA aromatic nitro compound and B are further mixed₂(OH)₄The reduction reaction is carried out to generate the corresponding on-DNA arylamine compound 3 in situ, the 3 is mixed with sodium nitrite, p-toluenesulfonic acid and triazo trimethyl silane, and the chemical reaction formula of the corresponding on-DNA aryl azide compound 4 is obtained by the in situ reaction.

FIG. 2A is a statistical chart of the conversion rate of the raw material on-DNA aryl azide compound 4 prepared in step 3 of the method of the present invention.

FIG. 2B is a graph of the conversion of the partially represented structural formula in FIG. 2A.

FIG. 3 is a chemical reaction formula of the method for preparing the corresponding on-DNA aryl phosphoramidate compound by mixing the on-DNA aryl azide compound 4 with trimethyl phosphite and carrying out in-situ reaction.

FIG. 4 is a graph showing the conversion rate of the representative structural formula of the corresponding on-DNA aryl phosphoramidate compound obtained by mixing the on-DNA aryl azide compound 4 with trimethyl phosphite and carrying out an in situ reaction (the conversion rate is based on the area of the TIC peak on LCMS-LTQ).

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.