CN110658163A - Method for monitoring reaction in synthesis of DNA coding compound - Google Patents
Method for monitoring reaction in synthesis of DNA coding compound Download PDFInfo
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- CN110658163A CN110658163A CN201910573027.2A CN201910573027A CN110658163A CN 110658163 A CN110658163 A CN 110658163A CN 201910573027 A CN201910573027 A CN 201910573027A CN 110658163 A CN110658163 A CN 110658163A
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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"
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- 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
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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
Abstract
The present invention provides a method for monitoring a reaction in synthesizing a DNA-encoding compound. The reaction monitoring method in synthesizing the DNA coding compound applies the fluorescence labeling technology to the synthesis of the DNA coding compound. The method can quickly, accurately and efficiently monitor the conversion degree of the reaction of the synthetic DNA coding compound mixed system.
Description
Technical Field
The invention relates in particular to a method for monitoring the reaction in the synthesis of DNA-encoding compounds.
Background
In drug development, especially new drug development, high-throughput screening for biological targets is one of the main means for rapidly obtaining lead compounds. However, traditional high throughput screening based on single molecules requires long time, large equipment investment, limited number of library compounds (millions), and the building of compound libraries requires decades of accumulation, limiting the efficiency and possibility of discovery of lead compounds. The recent emergence of DNA-encoded compound library synthesis technology, combining combinatorial chemistry and molecular biology techniques, with one DNA tag per compound at the molecular level, has enabled synthesis of libraries of compounds up to the billions in extremely short time. Moreover, compounds can be identified by a gene sequencing method, which greatly increases the size and synthesis efficiency of compound libraries, and becomes a trend of the next generation compound library screening technology, and starts to be widely applied in the foreign pharmaceutical industry, resulting in a lot of positive effects (Accounts of Chemical Research, 2014, 47, 1247-.
The DNA coding compound library can rapidly generate a giant compound library through combinatorial chemistry, and can screen out a lead compound with high flux, so that the screening of the lead compound becomes unprecedented rapidness and high efficiency. However, the generated compound library is a complex mixture system, and the currently commonly used LC-MS (liquid chromatography mass spectrometry)/HPLC chromatographic analysis methods, such as lingling, lixing, chenyanli, wenchao, hukefeng liquid chromatography mass spectrometry technology, are used for the analysis of small-molecule compounds in the complex mixture system [ J ]. chinese science: chemistry, 2017, 47 (12): 1379-. However, in the process of DNA coding compound library, a huge number of compounds (up to hundreds of thousands or even billions of compounds in mixed samples) are generated through a reaction step of mixing a plurality of samples, each compound has a different structure and a different molecular weight, and it is difficult for the conventional LC-MS to detect the molecular weight of each compound and the change of the molecular weight of each compound. Meanwhile, the polarities of different structures are different, and the conversion of the reaction cannot be detected by common monitoring methods such as HPLC and the like. Therefore, when synthesizing a DNA coding compound, the conversion degree of the target compound in the mixed system is difficult to accurately monitor due to the influence of LC-MS/HPLC instrument conditions, sample condition changes and the like.
Fluorescence labeling techniques involve the use of a fluorescent substance covalently bound or physically adsorbed to a moiety on the molecule to be studied to provide information about the subject being studied by virtue of its fluorescent properties. The method uses a commercial fluorescent reagent and a specific functional group modified at the tail end of DNA to quickly generate a stable fluorescent group, and obtains the actual concentration of the DNA coding compound by testing the fluorescent absorption of specific wavelength and comparing with a standard curve, thereby monitoring the consumption or the generated conversion degree of the specific functional group in the DNA coding compound. The method is not limited by instrument conditions and sample conditions, saves more time and labor and can accurately monitor the conversion degree of the compound in the mixed system.
Disclosure of Invention
To solve the above problems, the present invention provides a method for monitoring a reaction in synthesizing a DNA-encoding compound.
The technical scheme of the invention comprises the following steps:
a method for monitoring a reaction in the synthesis of a DNA-encoding compound, comprising the steps of:
(1) drawing a standard curve: preparing standard solutions with different concentrations by taking a DNA coding compound with a specific functional group at the tail end; adding a fluorescent reagent, measuring the fluorescence absorption value, and drawing a concentration-absorption standard curve;
(2) reaction monitoring is carried out: taking a mixed system of the DNA coding compound library with the tail end converted into the specific functional group and/or a mixed system after the DNA coding compound library with the tail end having the specific functional group is further reacted, and adding a fluorescent reagent; the fluorescence absorption value is measured, and the reaction conversion rate is calculated through a standard concentration-absorption curve.
The monitoring method as described above, wherein the DNA encoding compounds of step (1) and step (2) have the same skeleton structure;
"backbone structure" refers to a structure that is identical in structure in the small molecular moiety of each DNA-encoding compound in a library of DNA-encoding compounds;
and/or the DNA coding compound with the specific functional group at the end does not have fluorescence absorption or excitation under specific wavelength; the DNA coding compound library mixed system with the end converted into the specific functional group has no fluorescence absorption or excitation under specific wavelength;
and/or the mixed system obtained after the further reaction of the DNA coding compound library with the specific functional group at the tail end has no fluorescence absorption or excitation under specific wavelength;
and/or the specific functional group can react with a corresponding fluorescent reagent, and the product has fluorescence absorption or excitation at a specific wavelength.
As in the foregoing monitoring method, the specific functional groups described in step (1) and step (2) are primary amino, secondary amino, mercapto, carboxyl, hydroxyl, aldehyde, carbonyl, cyano; further, the specific functional group described in step (1) and step (2) is a primary amino group.
As the monitoring method described above, the specific functional groups described in the step (1) and the step (2) are amino groups, and the corresponding fluorescent reagents are 4-phenylspiro [ furan-2 (3H), 1-dihydroisobenzofuran ] -3, 3' -dione, 3- (2-furoyl) -quinoline-2-carboxaldehyde, o-phthalaldehyde, 2, 3-naphthalenedialdehyde, (4-carboxybenzoyl) -quinoline-2-carboxaldehyde, isothiocyanate, tetramethylrhodamine isothiocyanate, phenylisothiocyanate, dansyl chloride, 9-fluorenylmethylchloroformate, 4-fluoro-7-nitro-2, 1, 3-benzoxadiazole, 4-chloro-7-nitro-2, 1, 3-benzoxadiazole, 2-fluoro-4-naphthoxazolone, 1, 3-benzoxadiazole, and the like, 6-aminoquinoline-N-hydroxysuccinimide formate; further, the specific functional group described in step (1) and step (2) is a primary amino group, and the corresponding fluorescent reagent is 4-phenylspiro [ furan-2 (3H), 1-dihydroisobenzofuran ] -3, 3' -dione.
As the monitoring method, the specific functional group in the step (1) and the step (2) is carboxyl, and 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-benzooxadiazole, 9-aminopyrene-1, 4, 6, trisulfonic acid, and 8-aminopyrene-1, 3, 6-trisulfonic acid trisodium salt.
As for the monitoring method, the specific functional groups in the step (1) and the step (2) are hydroxyl, and the corresponding fluorescent reagents are 9-fluorenylmethoxycarbonyl chloride, 2-fluorenylsulfonyl chloride, dansyl chloride, anthracene-1-carbonyl nitrile and 1-ethoxy-4- (dichloro-S-triazine) naphthalene.
As the monitoring method, the specific functional groups in the step (1) and the step (2) are aldehyde groups and carbonyl groups, and the corresponding fluorescent reagents are dansyl hydrazide, 4- (N, N-dimethylaminosulfonyl) -7-hydrazino-2, 1, 3-benzooxadiazole, 4-hydrazino-7-nitro-2, 1, 3-benzooxadiazole (NBD-H) and 1, 3-cyclohexanedione.
As the monitoring method, the specific functional group in the step (1) and the step (2) is a sulfydryl group, and the corresponding fluorescent reagent is maleimide fluorescent reagent, iodoacetamide fluorescent reagent, aziridine fluorescent reagent or phthalic dialdehyde fluorescent reagent.
As for the monitoring method, the specific functional group in the step (1) and the step (2) is a cyano group, and the corresponding fluorescent reagents are boric acid group fluorescent reagents, salicylaldehyde fluorescent reagents, diphenylethanedione fluorescent reagents and acridine orange fluorescent reagents.
As for the monitoring method, the DNA coding compound library mixed system with the terminal converted into the specific functional group in the step (2) is a DNA coding compound library mixed system with the terminal azide group.
The fluorescence absorbance was measured at 388nm for excitation and 475nm for emission in steps (1) and (2) as described for the monitoring method described above.
As for the monitoring method described above, the range of the standard solution of different concentrations in step (1) is 0 to 1.0mmol/L, preferably 0 to 0.5 mm/L.
As for the monitoring method, after the fluorescent reagent corresponding to the specific functional group is added in the steps (1) and (2), the incubation is carried out for 5 to 60 minutes; preferably, incubation is for 10 minutes;
and/or the incubation temperature is 5-40 ℃; preferably, room temperature.
The monitoring method as described above, wherein the amount of the fluorescent reagent (amount of the substance) added in the steps (1) and (2) is 10 to 50 times the amount of the DNA-encoding compound, or 10 to 50 times the total concentration of the library of DNA-encoding compounds; preferably, the multiple is 20 times.
As for the monitoring method, the number of the standard solutions with different concentrations in the step (1) is an integer larger than 5.
"DNA-encoding compound having a specific functional group at the end" means a DNA-encoding compound having a specific functional group in a small molecule moiety;
"fluorescent reagent corresponding to a specific functional group" refers to a fluorescent reagent that can react with a specific functional group to generate a product having fluorescence absorption or excitation at a specific wavelength;
the "mixed system of DNA coding compound library whose terminal is converted into a specific functional group" means a mixed system of DNA coding compound library in which other functional groups are converted into specific functional groups during the reaction of the DNA coding compound library;
the "mixed system after further reaction of a library of DNA coding compounds having a specific functional group at the terminal" refers to a mixed system of libraries of DNA coding compounds in which the specific functional group is further reacted to generate another functional group during the reaction of the library of DNA coding compounds.
In the invention, "APTS" refers to trisodium salt of 8-aminopyrene-1, 3, 6-trisulfonate.
The invention has the following beneficial effects:
1. the invention can realize the accurate monitoring of the reaction process of the DNA coding compound, and the accuracy rate is equivalent to that of the liquid chromatogram-mass spectrum combination method.
2. The invention can avoid the detection of parameters in the aspects of molecular weight, molecular polarity and the like, and overcomes the defect that the traditional methods such as liquid chromatography-mass spectrometry, liquid phase and the like can not accurately distinguish the compound libraries for detecting the compound types up to hundreds of thousands or even hundreds of billions.
3. The monitoring method of the invention has short time consumption and convenient operation.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 shows a standard curve of DNA-NH2 solution absorption-concentration.
FIG. 2 shows the standard curve of DNA-NH2 solution absorption-concentration in the mixed system.
FIG. 3 is a standard curve of absorbance-concentration of a single DNA encoding compound.
FIG. 4 is a schematic of a single sample experimental procedure.
FIG. 5 is a schematic representation of the reaction sequence of single sample runs 1a to 1 b.
FIG. 6 is a graph of primary amine concentration versus time for the course of the reaction for a single sample 1 a-1 b.
FIG. 7 is a graph of the reaction process yield versus time for the single samples 1 a-1 b.
FIG. 8 is a schematic representation of the reaction process of single sample runs 1b to 1 c.
FIG. 9 is a graph of primary amine concentration versus time for the course of the reaction for a single sample 1 b-1 c.
FIG. 10 is a graph of reaction process yield versus time for the single samples 1 b-1 c.
FIG. 11 is a schematic diagram of the experimental procedure for mixing samples.
FIG. 12 is a schematic representation of the reaction process of the mixed sample experiments 2a to 2 b.
FIG. 13 is a graph of Bonn concentration versus time for the course of the reaction for mixed samples 2 a-2 b.
FIG. 14 is a graph of the reaction process yield versus time for mixed samples 2 a-2 b.
FIG. 15 is a schematic representation of the reaction process of the mixed sample experiments 2b to 2 c.
FIG. 16 Primary amine concentration versus time for the reaction process for Mixed samples 2b to 2 c.
FIG. 17 is a graph of the reaction process yield versus time for mixed samples 2 b-2 c.
FIG. 18 characterization of DNA-COOH by LC-MS.
FIG. 19 DNA-COOH solution absorption-concentration standard curve.
FIG. 20 shows a standard curve of DNA-COOH solution absorption-concentration in the mixed system.
FIG. 21 LC-MS characterization of the DNA-COOH reaction course in the mixed system.
FIG. 22 is a graph of carboxyl conversion versus time for a mixed system.
FIG. 23 is a schematic diagram of the reaction of compounds D to E.
Detailed Description
The process of the present invention is further illustrated below with examples of specific functional groups as primary amines. It is to be emphasized that: other amino, carboxyl, aldehyde, carbonyl, mercapto, cyano groups are also within the scope of the invention.
Example 1 plotting of concentration-absorption Standard Curve of DNA-NH2 solution
An initial 0.5mmol/L DNA-NH2 solution was prepared into 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50mmol/L standard solutions. A10-fold amount of the substance of the fluorescent reagent 4-phenylspiro [ furan-2 (3H), 1-dihydroisobenzofuran ] -3, 3' -dione in acetone (50mmol/L) was added and incubated at 5 ℃ for 10 minutes. Fluorescence absorption was measured at an excitation wavelength of 388nm and an emission wavelength of 475nm, and an absorption-concentration standard curve was plotted, as shown in FIG. 1.
Example 2 concentration-Absorbance calibration Curve of DNA-NH2 solution in Mixed System
DNA-NH2 solution and DNA-NH solution with initial concentration of 0.5mmol/L are mixed to prepare solution with DNA-NH2 ratio (mol) of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90%. A50-fold amount (amount of substance) of the fluorescent reagent in acetone (50mmol/L) was added and incubated at 40 ℃ for 5 minutes. Fluorescence absorption was measured at an excitation wavelength of 388nm and an emission wavelength of 475nm, and an absorption-concentration standard curve was plotted, as shown in FIG. 2.
Example 3 monitoring of the course of the reaction in a Complex System of libraries of DNA-encoding compounds
1. Standard curve was drawn with single DNA with similar backbone
Taking 1b (structural formula shown in figure 5) solution with initial concentration of 0.5mmol/L, and preparing into standard solution with concentration of 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50 mmol/L. A20-fold (amount of substance) acetone solution (50mmol/L) of the fluorescent reagent was added and incubated at room temperature for 10 minutes. And detecting fluorescence absorption at an excitation wavelength of 388nm and an emission wavelength of 475nm, and drawing an absorption-concentration standard curve. As shown in fig. 3
2. Single DNA reaction monitoring Process (see FIG. 4)
2.11 a to 1b reaction (see FIG. 5)
The reaction was started by dissolving 1a in sodium borate buffer (pH 9.4, concentration 250mmol/L) to prepare a 1mmol/L solution, adding tris (2-carboxyethyl) phosphine (100mmol/L in sodium borate buffer (pH 9.4, concentration 250mmol/L)) to a centrifuge tube at a molar ratio of 1: 100, and standing at room temperature. After the reaction was started, samples were taken at 0, 10, 20, 30, 45, 60, 90, 120, 180, 240, 600 minutes to detect LC-MS and fluorescence intensity. And substituting the measured fluorescence intensity value into a standard curve to obtain the primary amine concentration of the reaction system at the moment, and converting into the yield. The specific information is shown in table 1. The beran concentration of a single sample is plotted against time in table 1 as figure 6 and the yield of a single sample is plotted against time in table 1 as figure 7.
TABLE 1 Single sample 1a to 1b reaction information
2.21 b to 1c reaction (see FIG. 8)
Dissolving 1b in sodium borate buffer (pH 9.4, concentration 250mmol/L), adding benzoic acid (200mmol/L in N, N-dimethylacetamide), 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethylurea hexafluorophosphate (200mmol/L in N, N-dimethylacetamide), N, N-diisopropylethylamine (200mmol/L in N, N-dimethylacetamide) into a centrifuge tube according to the molar ratio of 1: 100, and placing at room temperature to start reaction. After the reaction was started, samples were taken at 0, 10, 20, 30, 45, 60, 90, 120, 180, 240, 600 minutes to detect LC-MS and fluorescence intensity. And substituting the measured fluorescence intensity value into a standard curve to obtain the primary amine concentration of the reaction system at the moment, and converting into the yield. The specific information is shown in Table 2. The concentration of beran in a single sample plotted according to Table 2 versus time is shown in FIG. 9, and the yield of a single sample plotted according to Table 2 versus time is shown in FIG. 10
TABLE 2 Single sample 1b to 1c reaction information
3. Reaction monitoring of Mixed samples (FIG. 11)
3.12 a to 2b reactions (FIG. 12)
The reaction was started by dissolving 2a in sodium borate buffer (pH 9.4, concentration 250mmol/L) and tris (2-carboxyethyl) phosphine (100mmol/L in sodium borate buffer (pH 9.4, concentration 250mmol/L)) in a molar ratio of 1: 100 into a centrifuge tube and placing at room temperature. After the reaction is started, samples are taken at 0, 10, 20, 30, 45, 60, 90, 120, 180, 240 and 600 minutes to detect the fluorescence intensity. And substituting the measured fluorescence intensity value into a standard curve to obtain the primary amine concentration of the reaction system at the moment, and converting into the yield. The specific information is shown in Table 3. The beran concentration of the mixed sample is plotted against time in accordance with table 2, as shown in fig. 13, and the yield of the mixed sample is plotted against time in accordance with table 2, as shown in fig. 14.
Table 3 mixed samples 2a to 2b reaction information
3.22b to 2c reaction (FIG. 15)
2b was dissolved in sodium borate buffer (pH 9.4, 250mmol/L), and added to a centrifuge tube at room temperature with benzoic acid (200mmol/L in N, N-dimethylacetamide), 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (200mmol/L in N, N-dimethylacetamide), N, N-diisopropylethylamine (200mmol/L in N, N-dimethylacetamide) in a molar ratio of 1: 100 to start the reaction. After the reaction is started, samples are taken at 0, 10, 20, 30, 45, 60, 90, 120, 180, 240 and 600 minutes to detect the fluorescence intensity. And substituting the measured fluorescence intensity value into a standard curve to obtain the primary amine concentration of the reaction system at the moment, and converting into the yield. The specific information is shown in Table 4. The beran concentration of the mixed sample is plotted against time in accordance with table 4 in fig. 16, and the yield of the mixed sample is plotted against time in accordance with table 2 in fig. 17.
Table 4 reaction information for mixed samples 2b to 2c
The above experimental results show that the fluorescence quantitative yield results are consistent with LC-MS for the detection of a single sample. The yield results for the mixed samples were consistent with the single sample. The fluorescent reagent can be used for detecting consumption and generation of primary amine of a mixed sample, and can accurately reflect the actual yield of the process.
Example 4 concentration-Absorbance calibration Curve of DNA-COOH solution
Step 1: DNA-COOH was dissolved in 250mmol/L phosphate buffer (pH 5.5) to prepare a 0.5mmol/L solution, 20 equivalents (amount of substance) of APTS stock solution (200mmol/L in water) and 300 equivalents of DMT (condensation reagent, 4- (4, 6-dimethoxytriazine) -4-methylmorpholine hydrochloride) aqueous solution (300mmol/L in water) were added and reacted at 45 ℃ for 12 hours (LC-MS detection reaction completed, and the LC-MS characterization result is shown in FIG. 18).
Step 2: precipitating the reaction product of step 1 with ethanol for 2 times to obtain 0.10mmol/L aqueous solution of carboxyl fluorescent marker C, diluting the aqueous solution into 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 solutions, measuring the fluorescence intensity of each concentration gradient by taking 20ul solution, and drawing a standard curve of the concentration and fluorescence intensity of carboxyl fluorescent marker C, as shown in FIG. 19.
Example 5 concentration-Absorbance calibration Curve of DNA-COOH solution in Mixed System
Step 1:
preparing DNA-COOH into a 0.5mmol/L solution (dissolved in 2500mmol/L phosphate buffer solution, pH is 5.5), adding 20 times of equivalent APTS stock solution (200mmol/L dissolved in water) and 300 times of equivalent DMT aqueous solution (300mmol/L), reacting at 45 ℃ for 12 hours, and performing LC-MS detection reaction, and then preparing into a 0.1mmol/L aqueous solution B through twice ethanol precipitation.
Preparing DNA-COOH into a 0.5mmol/L solution (dissolved in 2500mmol/L phosphate buffer solution, pH is 5.5), adding 20 times of an equivalent of phenylethylamine stock solution (200mmol/L dissolved in a mixed solvent prepared from 1 volume part of acetonitrile and 1 volume part of water), adding 300 times of an equivalent of DMT aqueous solution (300mmol/L), reacting at 45 ℃ for 12 hours, and performing LC-MS detection reaction, and performing ethanol precipitation twice to prepare a 0.1mmol/L aqueous solution C.
Step 2: the B/C solution was mixed with 0%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% of B content to give a mixed solution (0.1mmol/L DNA), and the content of the carboxyl fluorescent label in the mixed solution was measured against the standard curve of fluorescence intensity, as shown in FIG. 20.
Monitoring process of DNA-COOH reaction in the mixed system:
preparing DNA-COOH into a 0.5mmol/L solution (dissolved in 2500mmol/L phosphate buffer solution, pH 5.5), adding 20 times of equivalent phenylethylamine stock solution (200mmol/L dissolved in a mixed solvent prepared from 1 volume part of acetonitrile and 1 volume part of water), adding 300 times of equivalent DMT aqueous solution (300mmol/L), respectively taking 20nmol of DNA sample after 0.1, 0.5, 1.0, 2, 4, 8, 12, 16 and 24 hours from the beginning of the reaction, precipitating with ethanol twice, adding 20 times of equivalent APTS stock solution (200mmol/L dissolved in water) and 300 times of equivalent DMT aqueous solution (300mmol/L), reacting at 45 ℃ for 12 hours, and determining the fluorescence intensity of the reaction system by ethanol precipitation twice.
The measured fluorescence intensity was compared to the mixed sample standard curve to obtain the corresponding carboxyl content and compared to the LC-MS at the corresponding time point (see FIG. 21 for the LC-MS characterization results). The specific information is shown in Table 5. The DNA-COOH conversion in the mixed system plotted according to Table 5 as a function of time is shown in FIG. 22.
TABLE 5 DNA-COOH reaction information in the Mixed System
Example 6 monitoring of the course of the reaction in a Complex System of libraries of DNA-encoding compounds
The reaction process of compounds D to E is shown in FIG. 23. The monitoring method comprises the following steps:
1. preparing a carboxyl-containing DNA mixture into a 0.5mmol/L solution (dissolved in 2500mmol/L phosphate buffer solution, pH is 5.5), adding 20 times of equivalent of an amino reagent (which can be various amine reagent stock solutions) stock solution (200mmol/L dissolved in a mixed solvent prepared from 1 volume part of acetonitrile and 1 volume part of water), adding 300 times of equivalent of DMT aqueous solution (300mmol/L), reacting for 12 hours, precipitating with ethanol twice, adding 20 times of equivalent of APTS stock solution (200mmol/L dissolved in water) and 300 times of equivalent of DMT aqueous solution (300mmol/L), reacting for 12 hours at 45 ℃, and determining the fluorescence intensity of the reaction system through twice ethanol precipitation.
2. After the reaction is complete, the fluorescence intensity is measured and the carboxylic acid content is calculated from the standard curve.
The above experimental results show that the fluorescence quantitative yield results are consistent with LC-MS for the detection of a single sample. The yield results for the mixed samples were consistent with the single sample. The fluorescent reagent can be used for detecting the consumption and generation of carboxyl of a mixed sample, and can accurately reflect the actual yield of the process.
In conclusion, the invention can quickly and accurately realize the reaction monitoring of the DNA coding compound and has good application prospect.
Claims (15)
1. A method for monitoring a reaction in synthesizing a DNA-encoding compound, comprising: the method comprises the following operation steps:
(1) drawing a standard curve: preparing standard solutions with different concentrations by taking a DNA coding compound with a specific functional group at the tail end; adding a fluorescent reagent, measuring the fluorescence absorption value, and drawing a concentration-absorption standard curve;
(2) reaction monitoring is carried out: taking a mixed system of the DNA coding compound library with the tail end converted into the specific functional group and/or a mixed system after the DNA coding compound library with the tail end having the specific functional group is further reacted, and adding a fluorescent reagent; the fluorescence absorption value is measured, and the reaction conversion rate is calculated through a standard concentration-absorption curve.
2. The monitoring method according to claim 1, wherein: the DNA coding compounds in the step (1) and the step (2) have the same framework structure;
"backbone structure" refers to a structure that is identical in structure in the small molecular moiety of each DNA-encoding compound in a library of DNA-encoding compounds;
and/or the DNA coding compound with the specific functional group at the end does not have fluorescence absorption or excitation under specific wavelength; the DNA coding compound library mixed system with the end converted into the specific functional group has no fluorescence absorption or excitation under specific wavelength;
and/or the mixed system obtained after the further reaction of the DNA coding compound library with the specific functional group at the tail end has no fluorescence absorption or excitation under specific wavelength;
and/or the specific functional group can react with a corresponding fluorescent reagent, and the product has fluorescence absorption or excitation at a specific wavelength.
3. The monitoring method according to claim 1, wherein: the specific functional groups in the step (1) and the step (2) are primary amino, secondary amino, sulfydryl, carboxyl, hydroxyl, aldehyde group, carbonyl and cyano; further, the specific functional group described in step (1) and step (2) is a primary amino group.
4. The monitoring method according to claim 3, wherein: the specific functional group in the step (1) and the step (2) is amino, the corresponding fluorescent reagent is 4-phenyl spiro [ furan-2 (3H), 1-dihydroisobenzofuran ] -3, 3' -dione, 3- (2-furoyl) -quinoline-2-carboxaldehyde, o-phthalaldehyde, 2, 3-naphthaldehyde, (4-carboxybenzoyl) -quinoline-2-carboxaldehyde, isothiocyanate, tetramethylrhodamine isothiocyanate, benzene isothiocyanate, dansyl chloride, 9-fluorenemethylchloroformate, 4-fluoro-7-nitro-2, 1, 3-benzooxadiazole, 4-chloro-7-nitro-2, 1, 3-benzooxadiazole, 6-aminoquinoline-N-hydroxysuccinimide formate; further, the specific functional group described in step (1) and step (2) is a primary amino group, and the corresponding fluorescent reagent is 4-phenylspiro [ furan-2 (3H), 1-dihydroisobenzofuran ] -3, 3' -dione.
5. The monitoring method according to claim 3, wherein: the specific functional group in the step (1) and the step (2) is carboxyl, and 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-benzooxadiazole, 9-aminopyrene-1, 4, 6, trisulfonic acid, and 8-aminopyrene-1, 3, 6-trisulfonic acid trisodium salt.
6. The monitoring method according to claim 3, wherein: the specific functional group in the step (1) and the step (2) is hydroxyl, and the corresponding fluorescent reagent is 9-fluorenylmethoxycarbonyl chloride, 2-fluorenylsulfonyl chloride, dansyl chloride, anthracene-1-carbonyl nitrile, and 1-ethoxy-4- (dichloro-S-triazine) naphthalene.
7. The monitoring method according to claim 3, wherein: the specific functional groups in the step (1) and the step (2) are aldehyde groups and carbonyl groups, and the corresponding fluorescent reagents are dansyl hydrazide, 4- (N, N-dimethylaminosulfonyl) -7-hydrazino-2, 1, 3-benzooxadiazole, 4-hydrazino-7-nitro-2, 1, 3-benzooxadiazole (NBD-H) and 1, 3-cyclohexanedione.
8. The monitoring method according to claim 3, wherein: the specific functional group in the step (1) and the step (2) is sulfydryl, and the corresponding fluorescent reagent is maleimide fluorescent reagent, iodoacetamide fluorescent reagent, aziridine fluorescent reagent or phthalic dialdehyde fluorescent reagent.
9. The monitoring method according to claim 3, wherein: the specific functional group in the step (1) and the step (2) is a cyano group, and the corresponding fluorescent reagent is a boric acid group fluorescent reagent, a salicylaldehyde fluorescent reagent, a diphenylethanedione fluorescent reagent or an acridine orange fluorescent reagent.
10. The monitoring method according to claim 1, wherein: and (3) converting the tail end into a DNA coding compound library mixed system with a specific functional group in the step (2), wherein the tail end of the DNA coding compound library mixed system is a DNA coding compound library mixed system with an azide group.
11. The monitoring method according to claim 1, wherein: the fluorescence absorption values in step (1) and step (2) were measured at an excitation wavelength of 388nm and an emission wavelength of 475 nm.
12. The monitoring method according to claim 1, wherein: the range of the standard solution of different concentrations in step (1) is 0 to 1.0mmol/L, preferably 0 to 0.5 mm/L.
13. The monitoring method according to claim 1, wherein: after the fluorescent reagent corresponding to the specific functional group is added in the step (1) and the step (2), the incubation is carried out for 5 to 60 minutes; preferably, incubation is for 10 minutes;
and/or the incubation temperature is 5-40 ℃; preferably, room temperature.
14. The monitoring method according to claim 1, wherein: the adding amount of the fluorescent reagent in the step (1) and the step (2) is 10 to 50 times of the DNA coding compound, or 10 to 50 times of the total concentration of the DNA coding compound library; preferably, the multiple is 20 times.
15. The detection method according to claim 1, characterized in that: the number of the standard solutions with different concentrations in the step (1) is an integer larger than 5.
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