WO2020001560A1 - 一种合成dna编码化合物中的反应监测方法 - Google Patents

一种合成dna编码化合物中的反应监测方法 Download PDF

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WO2020001560A1
WO2020001560A1 PCT/CN2019/093377 CN2019093377W WO2020001560A1 WO 2020001560 A1 WO2020001560 A1 WO 2020001560A1 CN 2019093377 W CN2019093377 W CN 2019093377W WO 2020001560 A1 WO2020001560 A1 WO 2020001560A1
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dna
specific functional
monitoring method
group
functional group
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French (fr)
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李进
张帅
何窦
万金桥
刘观赛
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成都先导药物开发股份有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • 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
    • C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

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  • the invention particularly relates to a reaction monitoring method in a synthetic DNA-encoded compound.
  • compounds can be identified by gene sequencing, which has greatly increased the size and synthesis efficiency of compound libraries, and has become the trend of the next generation compound library screening technology. It has begun to be widely used in foreign pharmaceutical industries and has produced many positive effects ( Accounts of Chemical Research, 2014, 47, 1247-1255).
  • DNA-encoded compound libraries can quickly generate giant compound libraries through combinatorial chemistry, and lead compounds can be screened with high throughput, making the selection of lead compounds faster and more efficient than ever.
  • the resulting compound library is a complex mixture system, currently commonly used LC-MS (liquid chromatography mass spectrometry) / HPLC chromatography analysis methods, such as Yu Lingling, Li Xing, Chen Yanli, Wen Chao, Hu Kaifeng.
  • Fluorescence labeling technology refers to the use of some substances capable of emitting fluorescence to covalently bind or physically adsorb on a certain group of the molecule to be studied, and to use its fluorescence characteristics to provide information about the object under study.
  • the method uses a commercial fluorescent reagent and a specific functional group modified at the end of the DNA to quickly generate a stable fluorescent group. By measuring the fluorescence absorption at a specific wavelength and comparing it with a standard curve, the actual DNA-encoding compound concentration is obtained, thereby monitoring the DNA-encoding compound The degree of conversion of specific functional groups in the consumption or formation. This method is not restricted by instrument conditions and sample conditions, saves time and effort, and can more accurately monitor the degree of conversion of compounds in a mixed system.
  • the present invention provides a reaction monitoring method in synthesizing a DNA-encoded compound.
  • a reaction monitoring method for synthesizing a DNA-encoded compound includes the following operation steps: (1) drawing of a standard curve: taking a DNA-encoding compound having a specific functional group at the end, preparing standard solutions of different concentrations; adding a fluorescent reagent to measure fluorescence absorption Value, draw the concentration-absorption standard curve;
  • step (1) and step (2) have the same backbone structure
  • Skleton structure refers to a structure with the same structure in the small molecule structure portion of each DNA-encoding compound in the DNA-encoding compound library
  • the DNA-encoded compound having a specific functional group at the end has no fluorescence absorption or excitation at a specific wavelength;
  • the DNA-encoding compound library mixed system in which the end is converted to a specific functional group has no fluorescence absorption or excitation at a specific wavelength;
  • the mixed system after further reaction of the library of DNA-encoded compounds having specific functional groups at the ends has no fluorescence absorption or excitation at a specific wavelength
  • the specific functional group can react with the corresponding fluorescent reagent, and the product has fluorescence absorption or excitation at a specific wavelength.
  • the specific functional groups described in step (1) and step (2) are primary amino group, secondary amino group, mercapto group, carboxyl group, hydroxyl group, aldehyde group, carbonyl group, and cyano group; further, step (1) And the specific functional group described in step (2) is a primary amino group.
  • the specific functional group described in step (1) and step (2) is an amino group
  • the corresponding fluorescent reagent is 4-phenylspiro [furan-2 (3H), 1-dihydroisobenzo Furan] -3,3'-diketone, 3- (2-furanoformyl) -quinoline-2-carboxaldehyde, phthalaldehyde, 2,3-naphthalenedialdehyde, (4-carboxybenzoyl) -Quinoline-2-carboxaldehyde, isothiocyanate, tetramethylrhodamine isothiocyanate, benzene isothiocyanate, dansyl chloride, 9-fluorenyl methyl chloroformate, 4-fluoro -7-nitro-2,1,3-benzoxadiazole, 4-chloro-7-nitro-2,1,3-benzoxadiazole, 6-aminoquinoline-N-hydroxysuccinyl Imine formate
  • the specific functional group described in step (1) and step (2) is a carboxyl group
  • the corresponding fluorescent reagent is 1- (2-p-toluenesulfonate) ethyl-2-phenylimidazole ( 4,5) -9,10-phenanthrene, 4-N- (4-aminoethyl) piperazine-7-nitro-2,1,3-benzoxadiazole, 9-aminofluorene-1,4 , 6, trisulphonic acid, 8-aminophosphonium-1,3,6-trisulfonic acid trisodium salt.
  • the specific functional group described in step (1) and step (2) is a hydroxyl group
  • the corresponding fluorescent reagent is 9-fluorenylmethoxycarbonyl chloride, 2-fluorenylsulfonyl chloride, dansyl chloride, anthracene 1-carbonylnitrile, 1-ethoxy-4- (dichloro-S-triazine) naphthalene.
  • the specific functional groups described in steps (1) and (2) are aldehyde groups and carbonyl groups, and the corresponding fluorescent reagents are dansylhydrazide, 4- (N, N-dimethylaminosulfonyl) ) -7-hydrazino-2,1,3-benzoxadiazole, 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H), 1,3- Cyclohexanedione.
  • the specific functional group described in step (1) and step (2) is a thiol group
  • the corresponding fluorescent reagents are maleimide, iodoacetamide, aziridine, and phthalate Aldehyde fluorescent reagent.
  • the specific functional group described in step (1) and step (2) is a cyano group
  • the corresponding fluorescent reagent is a boric acid group, a salicylaldehyde group, a diphendione group, and an acridine orange.
  • Class fluorescent reagent is a boric acid group, a salicylaldehyde group, a diphendione group, and an acridine orange.
  • the mixed system of DNA-encoding compound libraries whose ends are converted into specific functional groups in step (2) is a mixed system of DNA-encoding compound libraries whose ends are azide groups.
  • the range of the standard solution with different concentrations in step (1) is 0 to 1.0 mmol / L, preferably 0 to 0.5 mm / L.
  • step (1) and step (2) after adding a fluorescent reagent corresponding to a specific functional group in step (1) and step (2), it needs to be incubated for 5 to 60 minutes; preferably, it is incubated for 10 minutes;
  • the incubation temperature is 5 to 40 ° C; preferably, it is room temperature.
  • the amount of fluorescent reagents added in steps (1) and (2) is 10 to 50 times the amount of DNA-encoded compounds, or 10 to 50 times the total concentration of the DNA-encoded compound library; Preferably, the multiple is 20 times.
  • the number of standard solutions of different concentrations in step (1) is an integer greater than five.
  • DNA-encoded compound with a specific functional group at the end means a DNA-encoded compound containing a specific functional group in a small molecule structure portion
  • Fluorescent reagent corresponding to a specific functional group refers to a fluorescent reagent capable of reacting with a specific functional group and a fluorescent reagent and generating a product that has a fluorescent absorption or excitation at a specific wavelength;
  • Mated system of DNA-encoding compound library whose end is converted into specific functional group refers to the mixed system of DNA-encoding compound library when other functional groups are converted into specific functional group during the reaction of the DNA-encoding compound library;
  • Mated system after further reaction of DNA-coding compound library with specific functional group at the end refers to the mixed system of DNA-coding compound library when specific functional group is further reacted to generate other functional groups during the reaction of DNA-coding compound library.
  • APTS means 8-aminofluorene-1,3,6-trisulfonic acid trisodium salt.
  • the present invention can achieve accurate monitoring of the reaction process of DNA-encoded compounds, and its accuracy is equivalent to that of the liquid chromatography-mass spectrometry method.
  • the invention can avoid the detection of parameters such as molecular weight and molecular polarity, and overcomes the disadvantages of traditional liquid chromatography-mass spectrometry, liquid phase and other methods that can not accurately distinguish the compound library of detection compounds up to hundreds of thousands or even tens of billions. .
  • the monitoring method of the present invention is short in time and convenient in operation.
  • Figure 3 Standard curve of absorption-concentration of a single DNA-encoding compound.
  • Figure 4 Schematic diagram of a single sample experiment.
  • FIG. 5 Schematic diagram of the reaction process of single sample experiment process 1a to 1b.
  • Fig. 8 Schematic diagram of the reaction process of single sample experiments 1b to 1c.
  • Fig. 9 The relationship between primary amine concentration and time during the reaction of single samples 1b to 1c.
  • FIG. 11 Schematic diagram of the mixed sample experiment process.
  • Figure 12 Schematic diagram of the reaction process of the mixed sample experimental process 2a to 2b.
  • Fig. 13 Curve of the relationship between the concentration and time of the reaction in the mixed samples 2a to 2b.
  • FIG. 15 Schematic diagram of the reaction process of the mixed sample experimental process 2b to 2c.
  • Figure 20 Standard curve of DNA-COOH solution absorption-concentration in mixed systems.
  • FIG. 23 Schematic diagram of the reaction process of compounds D to E.
  • a DNA-NH2 solution with an initial concentration of 0.5 mmol / L was prepared as a standard solution with a concentration of 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50 mmol / L.
  • the fluorescence absorption was detected at an excitation wavelength of 388 nm and an emission wavelength of 475 nm, and an absorption-concentration standard curve was drawn, as shown in FIG. 1.
  • DNA-NH2 solution and DNA-NH solution with an initial concentration of 0.5mmol / L mix and prepare the DNA-NH2 ratio (mol) is 10%, 20%, 30%, 40%, 50%, 60%, 70% , 80%, 90% solution.
  • a 50-fold (amount of substance) acetone solution (50 mmol / L) of the fluorescent reagent was added and incubated at 40 ° C for 5 minutes. The fluorescence absorption was detected at an excitation wavelength of 388 nm and an emission wavelength of 475 nm, and an absorption-concentration standard curve was drawn, as shown in FIG. 2.
  • the measured fluorescence intensity value was substituted into the standard curve to obtain the primary amine concentration of the reaction system at this time, and converted into a yield. See Table 1 for specific information.
  • the relationship curve between the concentration and time of a single sample drawn according to Table 1 is shown in FIG. 6, and the relationship curve between the yield and time of a single sample drawn according to Table 1 is shown in FIG. 7.
  • Dissolve 1b in sodium borate buffer (pH 9.4, concentration 250mmol / L), with benzoic acid (200mmol / L in N, N-dimethylacetamide), 2- (7-benzobenzotriazole ) -N, N, N ', N'-tetramethylurea hexafluorophosphate (200mmol / L in N, N-dimethylacetamide), N, N-diisopropylethylamine (200mmol / L L is dissolved in N, N-dimethylacetamide) was added to a centrifuge tube at a molar ratio of 1: 100: 100: 100, and the reaction was started at room temperature.
  • Dissolve 2b in sodium borate buffer (pH 9.4, concentration 250mmol / L), with benzoic acid (200mmol / L in N, N-dimethylacetamide), 2- (7-benzotriazole ) -N, N, N ', N'-tetramethylurea hexafluorophosphate (200mmol / L in N, N-dimethylacetamide), N, N-diisopropylethylamine (200mmol / L L is dissolved in N, N-dimethylacetamide) was added to a centrifuge tube at a molar ratio of 1: 100: 100: 100 and the reaction was started at room temperature.
  • Step 2 Precipitate the reaction product of Step 1 twice with ethanol to prepare a 0.10 mmol / L carboxyl fluorescent marker C aqueous solution, and dilute the aqueous solution to 0, 0.001, 0.005, 0.01, 0.02, 0.04, 0.06, 0.08, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.00 mmol / L solutions, take 20ul to measure the fluorescence intensity of each concentration gradient, and draw a standard curve of the concentration of carboxyl fluorescent marker C and the fluorescence intensity , As shown in Figure 19.
  • step 1
  • Step 2 The B / C solution is formulated into a mixed solution according to the B content of 0%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%. (0.1mmol / L DNA), and the standard curve of the carboxyl fluorescent label content and fluorescence intensity in the mixed solution was determined, as shown in FIG. 20.
  • Example 6 Monitoring of the reaction process in a complex system of DNA-encoded compound libraries
  • the reaction process of the compounds D to E is shown in FIG. 23.
  • the monitoring method is as follows:
  • the present invention can quickly and accurately implement reaction monitoring of DNA-encoded compounds, and has good application prospects.

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Abstract

一种合成DNA编码化合物中的反应监测方法。该合成DNA编码化合物中的反应监测方法是将荧光标记技术运用于合成DNA编码化合物中。该方法能快速，准确，高效的监测合成DNA编码化合物混合体系反应的转化程度。

Description

一种合成DNA编码化合物中的反应监测方法 技术领域
本发明具体涉及一种合成DNA编码化合物中的反应监测方法。
背景技术
在药物研发，尤其是新药研发中，针对生物靶标的高通量筛选是快速获得先导化合物的主要手段之一。然而，基于单个分子的传统高通量筛选所需时间长、设备投入巨大、库化合物数量有限(数百万)，且化合物库的建成需要数十年的积累，限制了先导化合物的发现效率与可能性。近年来出现的DNA编码化合物库合成技术，结合了组合化学和分子生物学技术，在分子水平上将每个化合物加上一个DNA标签，能在极短的时间内合成高达亿级的化合物库。而且化合物能够通过基因测序的方法进行识别，大幅度地增加了化合物库的大小和合成效率，成为下一代化合物库筛选技术的趋势，并开始在国外制药行业广泛应用，产生了诸多积极的效果(Accounts of Chemical Research,2014,47,1247-1255)。
DNA编码化合物库通过组合化学快速产生巨型化合物库，并且能高通量地筛选出先导化合物，使得先导化合物的筛选变得前所未有的快捷和高效。但是，产生的化合物库是一个复杂的混合物体系，目前常用的LC-MS(液相色谱质谱联用)/HPLC色谱分析方法，如余玲玲，李兴，陈燕丽，文超，胡凯锋.液质联用技术用于复杂混合物体系中小分子化合物的分析[J].中国科学：化学，2017,47(12):1379-1391.，综述了不同复杂混合物体系中小分子化合物使用LC-MS分析时，样品处理，数据采集，数据(前)处理等对分析结果的影响。然而，DNA编码化合物库过程中，经过多个样品混合的反应步骤产生巨大的化合物数量(混合样品中化合物种类高达几十万甚至上百亿)，每个化合物结构不一样，分子量不一样，常用的LC-MS难以检测出每一个化合物的分子量以及各化合物分子量的变化。同时不同的结构极性也不相同，通过HPLC等常用的监测方法也无法检测反应的转化。因此在合成DNA编码化合物时，受LC-MS/HPLC仪器条件，样品条件变化等影响，是难以准确监测混合体系目标化合物的转化程度。
荧光标记技术指利用一些能发射荧光的物质共价结合或物理吸附在所要研究分子的某个基团上，利用它的荧光特性来提供被研究对象的信息。本方法应用商业化的荧光试剂与DNA末端修饰的特定官能团快速生成稳定的荧 光基团，通过测试特定波长的荧光吸收，与标准曲线进行比较后得到实际的DNA编码化合物浓度，从而监测DNA编码化合物中特定官能团消耗或者生成的转化程度。本方法不受仪器条件，样品条件的制约，更省时省力，更能准确监测混合体系中化合物的转化程度。
发明内容
为解决上述问题，本发明提供了一种合成DNA编码化合物中的反应监测方法。
本发明的技术方案包括：
一种合成DNA编码化合物中的反应监测方法，它包括如下操作步骤：(1)标准曲线的绘制：取末端有特定官能团的DNA编码化合物，配制不同浓度的标准溶液；加入荧光试剂，测定荧光吸收值，绘制浓度-吸收标准曲线；
(2)进行反应监测：取末端转化为特定官能团的DNA编码化合物库混合体系和/或末端有特定官能团的DNA编码化合物库进一步反应后的混合体系，加入荧光试剂；测定荧光吸收值，通过标准浓度-吸收曲线，计算反应转化率。
如前述的监测方法，步骤(1)和步骤(2)所述DNA编码化合物具有相同的骨架结构；
“骨架结构”是指DNA编码化合物库中各DNA编码化合物小分子结构部分中结构一致的结构；
和/或，所述末端有特定官能团的DNA编码化合物在特定波长下无荧光吸收或激发；所述末端转化为特定官能团的DNA编码化合物库混合体系在特定波长下无荧光吸收或激发；
和/或，所述末端有特定官能团的DNA编码化合物库进一步反应后的混合体系在特定波长下无荧光吸收或激发；
和/或，所述的特定官能团可与相对应的荧光试剂反应，生成物在特定波长下具有荧光吸收或激发。
如前述的监测方法，步骤(1)和步骤(2)中所述的特定官能团为伯氨基、仲氨基、巯基、羧基、羟基、醛基、羰基、氰基；更进一步的，步骤(1)和步骤(2)中所述的特定官能团为伯氨基。
如前述的监测方法，步骤(1)和步骤(2)中所述的特定官能团为氨基，相对应的荧光试剂为4-苯基螺[呋喃-2(3H),1-二氢异苯并呋喃]-3,3'-二酮、3-(2-呋喃甲酰基)-喹啉-2-羰醛、邻苯二甲醛、2,3-萘二醛、(4-羧基苯甲酰基)-喹啉-2-羰醛、异硫氰酸酯、四甲基罗丹明异硫氰酸酯、苯异硫氰酸酯、 丹磺酰氯、9-芴甲基氯甲酸酯、4-氟-7-硝基-2,1,3-苯并恶二唑、4-氯-7-硝基-2,1,3-苯并恶二唑、6-氨基喹啉-N-羟基琥珀酰亚胺甲酸酯；更进一步的，步骤(1)和步骤(2)中所述的特定官能团为伯氨基，相对应的荧光试剂为4-苯基螺[呋喃-2(3H),1-二氢异苯并呋喃]-3,3'-二酮。
如前述的监测方法，步骤(1)和步骤(2)中所述的特定官能团为羧基，相对应的荧光试剂为1-(2-对甲苯磺酸酯)乙基-2-苯基咪唑(4，5)-9,10-菲、4-N-(4-氨基乙基)哌嗪-7-硝基-2,1,3-苯并恶二唑、9-氨基芘-1,4,6,三磺酸、8-氨基芘-1，3，6-三磺酸三钠盐。
如前述的监测方法，步骤(1)和步骤(2)中所述的特定官能团为羟基，相对应的荧光试剂为9-芴甲氧基羰酰氯、2-芴磺酰氯、丹磺酰氯、蒽-1-羰基腈、1-乙氧基-4-(二氯-S-三嗪)萘。
如前述的监测方法，步骤(1)和步骤(2)中所述的特定官能团为醛基、羰基，相对应的荧光试剂为丹磺酰肼、4-(N,N-二甲氨基磺酰)-7-肼基-2,1,3-苯并恶二唑、4-肼基-7-硝基-2,1,3-苯并恶二唑(NBD-H)、1,3-环己二酮。
如前述的监测方法，步骤(1)和步骤(2)中所述的特定官能团为巯基，相对应的荧光试剂为马来酰亚胺类、碘乙酰胺类、氮丙啶类、邻苯二醛类荧光试剂。
如前述的监测方法，步骤(1)和步骤(2)中所述的特定官能团为氰基，相对应的荧光试剂为硼酸基团类、水杨醛基类、二苯乙二酮类、吖啶橙类荧光试剂。
如前述的监测方法，步骤(2)中所述末端转化为特定官能团的DNA编码化合物库混合体系为末端为叠氮基团的DNA编码化合物库混合体系。
如前述的监测方法，步骤(1)和步骤(2)中荧光吸收值在激发波长＝388nm，发射波长＝475nm下进行测定。
如前述的监测方法，步骤(1)中的不同浓度的标准溶液范围为0至1.0mmol/L，优选地，为0至0.5mm/L。
如前述的监测方法，步骤(1)和步骤(2)中加入与特定官能团相对应的荧光试剂后，还需孵育5至60分钟；优选地，孵育10分钟；
和/或，孵育的温度为5～40℃；优选地，为室温。
如前述的监测方法，步骤(1)和步骤(2)中荧光试剂的加入量(物质的量)为DNA编码化合物的10到50倍，或者为DNA编码化合物库总浓度的10到50倍；优选地，所述倍数为20倍。
如前述的监测方法，步骤(1)中不同浓度的标准溶液个数为大于5的整 数。
“末端有特定官能团的DNA编码化合物”是指小分子结构部分含有特定的官能团的DNA编码化合物；
“与特定官能团相对应的荧光试剂”是指特定官能团与荧光试剂能发生反应，生成的产物在特定波长下具有荧光吸收或激发的荧光试剂；
“末端转化为特定官能团的DNA编码化合物库混合体系”是指DNA编码化合物库反应过程中，其它官能团转化为特定官能团时的DNA编码化合物库混合体系；
“末端有特定官能团的DNA编码化合物库进一步反应后的混合体系”是指DNA编码化合物库反应过程中，特定官能团进一步反应产生其它官能团时的DNA编码化合物库混合体系。
本发明中“APTS”是指8-氨基芘-1，3，6-三磺酸三钠盐。
本发明具有如下有益效果：
1.本发明可以实现DNA编码化合物反应过程的精准监控，其准确率和液相色谱-质谱联用方法的准确率相当。
2.本发明可以避免对分子量、分子极性等方面参数的检测，克服了传统的液质联用、液相等方法不能准确区分检测化合物种类高达几十万甚至上百亿的化合物库的弊端。
3.本发明的监测方法耗时短，操作方便。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1  DNA-NH2溶液吸收-浓度标准曲线。
图2  混合体系中DNA-NH2溶液吸收-浓度标准曲线。
图3  单一DNA编码化合物吸收-浓度标准曲线。
图4  单一样品实验过程示意图。
图5  单一样品实验过程1a至1b反应过程示意图。
图6  单一样品1a至1b反应过程伯胺浓度与时间关系曲线。
图7  单一样品1a至1b反应过程产率与时间关系曲线。
图8  单一样品实验过程1b至1c反应过程示意图。
图9  单一样品1b至1c反应过程伯胺浓度与时间关系曲线。
图10 单一样品1b至1c反应过程产率与时间关系曲线。
图11 混合样品实验过程示意图。
图12 混合样品实验过程2a至2b反应过程示意图。
图13 混合样品2a至2b反应过程伯安浓度与时间关系曲线。
图14 混合样品2a至2b反应过程产率与时间关系曲线。
图15 混合样品实验过程2b至2c反应过程示意图。
图16 混合样品2b至2c反应过程伯胺浓度与时间关系曲线。
图17 混合样品2b至2c反应过程产率与时间关系曲线。
图18 DNA-COOH制备LC-MS表征结果。
图19 DNA-COOH溶液吸收-浓度标准曲线。
图20 混合体系中DNA-COOH溶液吸收-浓度标准曲线。
图21 混合体系中DNA-COOH反应过程的LC-MS表征结果。
图22 混合体系中羧基转化率与时间关系曲线。
图23 化合物D至E反应过程示意图。
具体实施方式
以下以特定官能团为伯胺的实例，对本发明的方法进行进一步说明。需要强调的是：以其它氨基、羧基、醛基、羰基、巯基、氰基为特定官能团的情形仍然在本发明的保护范围之内。
实施例1、DNA-NH2溶液的浓度-吸收标准曲线的绘制
取初始浓度为0.5mmol/L的DNA-NH2溶液，配制成浓度为0.05、0.10、0.15、0.20、0.25、0.30、0.35、0.40、0.45、0.50mmol/L的标准溶液。加入10倍物质的量的荧光试剂4-苯基螺[呋喃-2(3H),1-二氢异苯并呋喃]-3,3'-二酮的丙酮溶液(50mmol/L)，5℃孵育10分钟。在激发波长388nm和发射波长475nm下检测荧光吸收，绘制吸收-浓度标准曲线，如图1所示。
实施例2、混合体系中DNA-NH2溶液的浓度-吸收标准曲线的绘制
取初始浓度为0.5mmol/L的DNA-NH2溶液和DNA-NH溶液，混合配制成DNA-NH2比例(mol)为10％、20％、30％、40％、50％、60％、70％、80％、90％的溶液。加入50倍(物质的量)的荧光试剂丙酮溶液(50mmol/L)，40℃孵育5分钟。在激发波长388nm和发射波长475nm下检测荧光吸收，绘制吸收-浓度标准曲线，如图2所示。
实施例3、DNA编码化合物库复杂体系中反应过程监测
1.用带相似骨架的单一DNA绘制标准曲线
取初始浓度为0.5mmol/L的1b(结构式如图5所示)溶液，配制成浓度为0.05、0.10、0.15、0.20、0.25、0.30、0.35、0.40、0.45、0.50mmol/L的标准溶液。加入20倍(物质的量)的荧光试剂丙酮溶液(50mmol/L)，室温孵育10分钟。在激发波长388nm和发射波长475nm下检测荧光吸收，绘制吸收-浓度标准曲线。如图3所示
2.单一DNA反应监测过程(如图4)
2.1 1a至1b反应(如图5)
将1a溶于硼酸钠缓冲液(pH＝9.4，浓度250mmol/L)中，配制为1mmol/L的溶液，与三(2-羧乙基)膦(100mmol/L溶于硼酸钠缓冲液(pH＝9.4,浓度250mmol/L))按照摩尔比1:100加入离心管中，置于室温开始反应。反应开始后，0、10、20、30、45、60、90、120、180、240、600分钟取样检测LC-MS和荧光强度。将测得的荧光强度值代入标准曲线，得到此时反应体系的伯胺浓度，并换算成产率。具体信息见表1。根据表1绘制的单一样品伯安浓度与时间关系曲线如图6，根据表1绘制的单一样品产率与时间关系曲线如图7。
表1 单一样品1a至1b反应信息
Figure PCTCN2019093377-appb-000001
2.2 1b至1c反应(如图8)
将1b溶于硼酸钠缓冲液(pH＝9.4，浓度250mmol/L)，与苯甲酸(200mmol/L溶于N,N-二甲基乙酰胺)、2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(200mmol/L溶于N,N-二甲基乙酰胺)、N,N-二异丙基乙胺(200mmol/L溶于N,N-二甲基乙酰胺)按照摩尔比1:100:100:100加入到离心管中，置于室温开始反应。反应开始后，0、10、20、30、45、60、90、120、180、240、600分钟取样检测LC-MS和荧光强度。将测得的荧光强度值代入标准曲线，得到此时反应体系的伯胺浓度，并换算成产率。具体信息见表2。根据表2绘制的单一样品伯安浓度与时间关系曲线如图9，根据表2绘制的单一样品产率与时间关系曲线如图10
表2 单一样品1b至1c反应信息
Figure PCTCN2019093377-appb-000002
3.混合样品的反应监测过程(图11)
3.1 2a至2b反应(图12)
将2a溶于硼酸钠缓冲液(pH＝9.4，浓度250mmol/L)，与三(2-羧乙基)膦(100mmol/L溶于硼酸钠缓冲液(pH＝9.4,浓度250mmol/L))按照摩尔比1:100加入到离心管中，置于室温开始反应。反应开始后，0、10、20、30、45、60、90、120、180、240、600分钟取样检测荧光强度。将测得的荧光强度值代入标准曲线，得到此时反应体系的伯胺浓度，并换算成产率。具体信息见表3。根据表2绘制的混合样品伯安浓度与时间关系曲线如图13，根据表2绘制的混合样品产率与时间关系曲线如图14。
表3 混合样品2a至2b反应信息
Figure PCTCN2019093377-appb-000003
3.2 2b至2c反应(图15)
将2b溶于硼酸钠缓冲液(pH＝9.4，浓度250mmol/L)，与苯甲酸(200mmol/L溶于N,N-二甲基乙酰胺)、2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(200mmol/L溶于N,N-二甲基乙酰胺)、N，N-二异丙基乙胺(200mmol/L溶于N,N-二甲基乙酰胺)按照摩尔比1:100:100:100加入到离心管中置于室温开始反应。反应开始后，0、10、20、30、45、60、90、120、180、240、600分钟取样检测荧光强度。将测得的荧光强度值代入标准曲线，得到此时反应体系的伯胺浓度，并换算成产率。具体信息见表4。根据表4绘制的混合样品伯安浓度与时间关系曲线如图16，根据表2绘制的混合样品产率与时间关系曲线如图17。
表4 混合样品2b至2c反应信息
Figure PCTCN2019093377-appb-000004
以上实验结果表明，对于单一样品的检测，荧光定量产率结果与LC-MS一致。混合样品的产率结果与单一样品一致。荧光试剂可以用于混合样品的伯胺消耗与生成的检测，并能够准确反映过程的实际收率。
实施例4、DNA-COOH溶液的浓度-吸收标准曲线的绘制
步骤1：取DNA-COOH配成0.5mmol/L溶液(溶于250mmol/L的磷酸缓冲液，pH＝5.5)，加入20倍当量(物质的量)的APTS储备液(200mmol/L溶于水)，300倍当量的DMT(缩合试剂，4-(4,6-二甲氧基三嗪)-4-甲基吗啉盐酸盐)水溶液(300mmol/L溶于水)，45℃反应12小时(LC-MS检测反应完全，LC-MS表征结果见图18)。
步骤2：将步骤1的反应产物乙醇沉淀2次后配成0.10mmol/L的羧基荧光标记物C水溶液，并将该水溶液稀释成0、0.001、0.005、0.01、0.02、0.04、0.06、0.08、0.10、0.20、0.30、0.40、0.50、0.60、0.70、0.80、0.90、1.00mmol/L的溶液，取20ul测定各浓度梯度的荧光强度，并绘制羧基荧光标记物C的浓度与荧光强度的标准曲线，如图19。
实施例5、混合体系中DNA-COOH溶液的浓度-吸收标准曲线的绘制
步骤1：
取DNA-COOH配成0.5mmol/L溶液(溶于2500mmol/L的磷酸缓冲液，pH＝5.5)，加入20倍当量的APTS储备液(200mmol/L溶于水)，300倍当量的DMT水溶液(300mmol/L)，45℃反应12小时，LC-MS检测反应完全后经过两次乙醇沉淀配成0.1mmol/L水溶液B。
取DNA-COOH配成0.5mmol/L溶液(溶于2500mmol/L的磷酸缓冲液，pH＝5.5)，加入20倍当量的苯乙胺储备液(200mmol/L，溶于混合溶剂中，混合溶剂由1体积份的乙腈和1体积份的水配制而成)，300倍当量的DMT水溶液(300mmol/L),45℃反应12小时，LC-MS检测反应完全后经过两次乙醇沉淀配成0.1mmol/L水溶液C。
步骤2：将B/C溶液按照B含量0％，5％，10％，20％，30％，40％，50％，60％，70％，80％，90％，100％配成混合溶液(0.1mmol/L DNA)，并测定 混合溶液中羧基荧光标记物含量与荧光强度的标准曲线，如图20。
混合体系中DNA-COOH反应监测过程：
取DNA-COOH配成0.5mmol/L溶液(溶于2500mmol/L的磷酸缓冲液，pH＝5.5)，加入20倍当量的苯乙胺储备液(200mmol/L，溶于混合溶剂中，混合溶剂由1体积份的乙腈和1体积份的水配制而成)，300倍当量的DMT水溶液(300mmol/L),分别于反应开始的0.1、0.5、1.0、2、4、8、12、16、24小时后取20nmolDNA样品，经两次乙醇沉淀后，加入20倍当量的APTS储备液(200mmol/L，溶于水)，300倍当量的DMT水溶液(300mmol/L),45℃反应12小时，再经过两次乙醇沉淀测定反应体系的荧光强度。
将测定的荧光强度与混合样品标准曲线比较，得到对应的羧基含量，并与相应时间点的LC-MS对比(LC-MS表征结果见图21)。具体信息见表5。根据表5绘制的混合体系中DNA-COOH转化率与时间关系曲线如图22。
表5 混合体系中DNA-COOH反应信息
Figure PCTCN2019093377-appb-000005
实施例6、DNA编码化合物库复杂体系中反应过程监测
化合物D至E反应过程如图23所示。监测方法如下：
1.取含有羧基的DNA混合物，配成0.5mmol/L溶液(溶于2500mmol/L的磷酸缓冲液，pH＝5.5)，加入20倍当量的氨基试剂(可以是各种胺试剂储备液)储备液(200mmol/L，溶于混合溶剂中，混合溶剂由1体积份的乙腈和1体积份的水配制而成)，300倍当量的DMT水溶液(300mmol/L),反应12小时后，经两次乙醇沉淀后，加入20倍当量的APTS储备液(200mmol/L，溶于水)，300倍当量的DMT水溶液(300mmol/L),45℃反应12小时，再经过两次乙醇沉淀测定反应体系的荧光强度。
2.反应结束后测定荧光强度，并由标准曲线计算羧酸含量。
以上实验结果表明，对于单一样品的检测，荧光定量产率结果与LC-MS一致。混合样品的产率结果与单一样品一致。荧光试剂可以用于混合样品的羧基消耗与生成的检测，并能够准确反映过程的实际收率。
综上，本发明可以快速、准确地实现DNA编码化合物的反应监测，具有良好的应用前景。

Claims (15)

  1. 一种合成DNA编码化合物中的反应监测方法，其特征在于：它包括如下操作步骤：
    (1)标准曲线的绘制：取末端有特定官能团的DNA编码化合物，配制不同浓度的标准溶液；加入荧光试剂，测定荧光吸收值，绘制浓度-吸收标准曲线；
    (2)进行反应监测：取末端转化为特定官能团的DNA编码化合物库混合体系和/或末端有特定官能团的DNA编码化合物库进一步反应后的混合体系，加入荧光试剂；测定荧光吸收值，通过标准浓度-吸收曲线，计算反应转化率。
  2. 根据权利要求1所述的监测方法，其特征在于：步骤(1)和步骤(2)所述DNA编码化合物具有相同的骨架结构；
    “骨架结构”是指DNA编码化合物库中各DNA编码化合物小分子结构部分中结构一致的结构；
    和/或，所述末端有特定官能团的DNA编码化合物在特定波长下无荧光吸收或激发；所述末端转化为特定官能团的DNA编码化合物库混合体系在特定波长下无荧光吸收或激发；
    和/或，所述末端有特定官能团的DNA编码化合物库进一步反应后的混合体系在特定波长下无荧光吸收或激发；
    和/或，所述的特定官能团可与相对应的荧光试剂反应，生成物在特定波长下具有荧光吸收或激发。
  3. 根据权利要求1所述的监测方法，其特征在于：步骤(1)和步骤(2)中所述的特定官能团为伯氨基、仲氨基、巯基、羧基、羟基、醛基、羰基、氰基；更进一步的，步骤(1)和步骤(2)中所述的特定官能团为伯氨基。
  4. 根据权利要求3所述的监测方法，其特征在于：步骤(1)和步骤(2)中所述的特定官能团为氨基，相对应的荧光试剂为4-苯基螺[呋喃-2(3H),1-二氢异苯并呋喃]-3,3'-二酮、3-(2-呋喃甲酰基)-喹啉-2-羰醛、邻苯二甲醛、2,3-萘二醛、(4-羧基苯甲酰基)-喹啉-2-羰醛、异硫氰酸酯、四甲基罗丹明异硫氰酸酯、苯异硫氰酸酯、丹磺酰氯、9-芴甲基氯甲酸酯、4-氟-7-硝基-2,1,3-苯并恶二唑、4-氯-7-硝基-2,1,3-苯并恶二唑、6-氨基喹啉-N-羟基琥珀酰亚胺甲酸酯；更进一步的，步骤(1)和步骤(2)中所述的特定官能团为伯氨基，相对应的荧光试剂为4-苯基螺[呋喃-2(3H),1-二氢异苯并呋喃]-3,3'-二酮。
  5. 根据权利要求3所述的监测方法，其特征在于：步骤(1)和步骤(2)中所述的特定官能团为羧基，相对应的荧光试剂为1-(2-对甲苯磺酸酯)乙 基-2-苯基咪唑(4，5)-9,10-菲、4-N-(4-氨基乙基)哌嗪-7-硝基-2,1,3-苯并恶二唑、9-氨基芘-1,4,6,三磺酸、8-氨基芘-1，3，6-三磺酸三钠盐。
  6. 根据权利要求3所述的监测方法，其特征在于：步骤(1)和步骤(2)中所述的特定官能团为羟基，相对应的荧光试剂为9-芴甲氧基羰酰氯、2-芴磺酰氯、丹磺酰氯、蒽-1-羰基腈、1-乙氧基-4-(二氯-S-三嗪)萘。
  7. 根据权利要求3所述的监测方法，其特征在于：步骤(1)和步骤(2)中所述的特定官能团为醛基、羰基，相对应的荧光试剂为丹磺酰肼、4-(N,N-二甲氨基磺酰)-7-肼基-2,1,3-苯并恶二唑、4-肼基-7-硝基-2,1,3-苯并恶二唑(NBD-H)、1,3-环己二酮。
  8. 根据权利要求3所述的监测方法，其特征在于：步骤(1)和步骤(2)中所述的特定官能团为巯基，相对应的荧光试剂为马来酰亚胺类、碘乙酰胺类、氮丙啶类、邻苯二醛类荧光试剂。
  9. 根据权利要求3所述的监测方法，其特征在于：步骤(1)和步骤(2)中所述的特定官能团为氰基，相对应的荧光试剂为硼酸基团类、水杨醛基类、二苯乙二酮类、吖啶橙类荧光试剂。
  10. 根据权利要求1所述的监测方法，其特征在于：步骤(2)中所述末端转化为特定官能团的DNA编码化合物库混合体系为末端为叠氮基团的DNA编码化合物库混合体系。
  11. 根据权利要求1所述的监测方法，其特征在于：步骤(1)和步骤(2)中荧光吸收值在激发波长＝388nm，发射波长＝475nm下进行测定。
  12. 根据权利要求1所述的监测方法，其特征在于：步骤(1)中的不同浓度的标准溶液范围为0至1.0mmol/L，优选地，为0至0.5mm/L。
  13. 根据权利要求1所述的监测方法，其特征在于：步骤(1)和步骤(2)中加入与特定官能团相对应的荧光试剂后，还需孵育5至60分钟；优选地，孵育10分钟；
    和/或，孵育的温度为5～40℃；优选地，为室温。
  14. 根据权利要求1所述的监测方法，其特征在于：步骤(1)和步骤(2)中荧光试剂的加入量为DNA编码化合物的10到50倍，或者为DNA编码化合物库总浓度的10到50倍；优选地，所述倍数为20倍。
  15. 根据权利要求1所述的检测方法，其特征在于：步骤(1)中不同浓度的标准溶液个数为大于5的整数。
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