Detection Method of 2-Cyanoethyl-N,N,N',N'-Tetraisopropyl Phosphorodiamidite

Abstract

The present invention provides a method for detecting 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite, the method comprising the steps of: (1) pre-treating a sample to be tested to obtain a test solution, wherein the pre-treatment causes the 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite in the sample to be tested to be derivatized into a compound shown in Formula I, (2) detecting the derivative product of 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite in the test solution by high performance liquid chromatography, obtaining the content of the derivative product in the test solution by an external standard method, and (3) calculating the content of 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite in the test sample. The detection method of the present invention does not require the use of NMR detection means, is easy to implement, has good repeatability and high accuracy. #imgabs0#

Description

Technical Field

[0001] The present invention relates to a compound analysis technology, in particular to a detection method for 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite. Background Art

[0002] 2-Cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite is a phosphoramidite reagent that is widely used in the synthesis of oligonucleotides. The main challenge in the chemical synthesis of oligonucleotides is to activate 3'-OH (or 5'-OH) so that the next nucleic acid unit can be added to the oligonucleotide chain one by one under relatively mild conditions. Historically, scientists have tried to use many different activating groups. After continuous optimization, a synthesis method using phosphoramidite as the 3'-OH activating group was developed in the early 1980s, and solid-phase synthesis and automated synthesis were successfully achieved.

[0003] 2-Cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite is unstable, sensitive to air and moisture, easily oxidized or hydrolyzed, and expensive, so researchers often prepare this phosphorus reagent on site, store it in solution, and then use it in feed to ensure the quality of the phosphorus reagent, which has a greater cost advantage. Usually, the solution of the phosphorus reagent prepared by researchers themselves needs to be accurately measured, because when synthesizing oligonucleotide monomers, the content of the phosphorus reagent needs to be accurately measured to calculate the feed ratio. Once the phosphorus reagent is used in excess, impurities will be generated during monomer synthesis, thereby affecting the quality of the oligonucleotide monomer. At present, there are very few reports on the detection method of phosphorus reagent content in solution. Most of them use NMR as a detection means, and calculate the content of phosphorus reagent by using the ratio of the characteristic peak area of ​​the analyte and the internal standard. For example, CN106132973A, CN103087096A, CN11123392, Large-scale synthesis of high purity "Phosreagent" useful for oligonucleotide therapeutics, R. Gukathasan et al., Journal of Organometallic Chemistry 690 (2005) 2603-2607, all disclose the method of detecting phosphorus reagent content in solution by using NMR as a detection means. 31 PNMR determines the content of phosphorodiamidite, but NMR itself is expensive and has high maintenance costs. Many pharmaceutical companies are not equipped with NMR and cannot use NMR to detect the content of phosphorus reagents. In addition, 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite itself has almost no ultraviolet absorption and is unstable to air and water, so it cannot be detected by conventional reversed-phase liquid chromatography.

[0004] Therefore, there is an urgent need to develop an analytical method that is easy to implement, has good repeatability and high accuracy for the detection of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite, for example, to detect the content of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite in a sample. Summary of the invention

[0005] In view of the defects of the detection method of 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite in the prior art, such as high cost and difficult operation, the present invention provides a simple, rapid and easy-to-implement detection method of 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite, which solves the technical problem that conventional liquid chromatography is difficult to directly detect phosphorus reagents. To achieve the purpose of the present invention, the present invention adopts the following technical scheme:

[0006] A method for detecting 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite, the method comprising the following steps:

[0007] (1) pre-treating the sample to be tested to obtain a test solution, wherein the pre-treatment causes 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite in the sample to be tested to be derivatized into a compound shown in Formula I,

[0008]

[0009] wherein R1, R2, R3, R4, R5, R6, and R7 are each independently hydrogen, halogen, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 alkoxy, phenoxy, nitro, cyano, or amide,

[0010] (2) using high performance liquid chromatography to detect the derivative product of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite in the test solution, and obtaining the concentration of the derivative product in the test solution by an external standard method,

[0011] (3) Calculate the content of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite in the sample to be tested.

[0012] In another preferred embodiment, the naphthylmethyl group in Formula I is selected from the following groups:

[0013]

[0014] The * represents a connection point.

[0015] In another preferred embodiment, the naphthylmethyl group in Formula I is selected from The * represents a connection point.

[0016] In another preferred embodiment, the pretreatment of the sample to be tested in step (1) includes the step of: mixing the sample to be tested with an activator and a compound shown in formula III, so that the 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite shown in formula II in the sample to be tested reacts with the compound shown in formula III in the presence of the activator to generate a compound shown in formula I, and the reaction formula is as follows:

[0017]

[0018] In another preferred embodiment, the activator is selected from 4,5-dicyanoimidazole.

[0019] In another preferred embodiment, in the reaction system, the molar ratio of the compound represented by formula III to the compound represented by formula II is 2 to 3:1.

[0020] In another preferred embodiment, in the reaction system, the molar ratio of the activator to the compound represented by formula II is 1 to 3:1.

[0021] In another preferred embodiment, the reaction temperature of the reaction system is 4-40°C.

[0022] In another preferred embodiment, in the high performance liquid chromatography detection, the detection wavelength used is 280nm±4nm; the chromatographic column used is a C8 column or a C18 column; the mobile phase consists of an ammonium acetate solution and acetonitrile, with an aqueous ammonium acetate solution as mobile phase A and acetonitrile as mobile phase B, and the gradient elution program is: 0-3min, 10% B; 3-10min, 10% B→50% B; 10-20min, 50% B→90% B; 20-28min, 90% B; 28-35min, 10% B, wherein the concentration of the aqueous ammonium acetate solution is 5mM-50mM / L.

[0023] In another preferred embodiment, the flow rate of the mobile phase is 0.2-2.0 mL / min.

[0024] In another preferred embodiment, the injection volume is 5 to 20 uL.

[0025] In another preferred embodiment, the detection method comprises preparing a standard solution containing the compound represented by formula I before high performance liquid chromatography detection, and the preparation method of the standard solution is:

[0026] (1) Preparation of linear stock solution: Accurately weigh 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite derivative reference substance into a volumetric flask, dilute to volume with acetonitrile to prepare a 6.0-9.0 mg / mL stock solution;

[0027] (2) Dilute the stock solution into 5 to 8 standard solutions with concentrations ranging from 0.2 to 1.5 mg / mL.

[0028] In another preferred embodiment, the method for obtaining the concentration of the derivative product in the test solution by the external standard method in step (2) comprises the following steps:

[0029] (a) injecting a standard solution containing the compound of formula I into a liquid chromatograph to obtain a liquid chromatogram of the standard solution, performing a linear regression with concentration as the abscissa and peak area as the ordinate to obtain a linear regression equation; and

[0030] (b) diluting the test solution obtained by the pretreatment and injecting it into a liquid chromatograph, obtaining a liquid chromatogram of the test solution under the same chromatographic conditions as in step (a), qualitatively analyzing the derivatives therein by retention time, and calculating the concentration of the compound represented by formula I in the test solution by peak area according to the linear regression equation.

[0031] In another preferred embodiment, in step (b), the test solution is diluted 10 to 50 times, and the concentration of the compound of formula I in the diluted solution is 0.3 to 1.2 mg / mL.

[0032] In another preferred embodiment, the content of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite in the sample to be tested is calculated in step (3) according to the following formula 1:

[0033]

[0034] Where: C 样 is the mass percentage content of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite in the sample to be tested, %;

[0035] C I is the concentration of the compound of formula I in the test solution,

[0036] V is the dilution factor of the test solution,

[0037] m 样 is the weight of the sample to be tested,

[0038] 301.41 is the molar mass of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite,

[0039] M I is the molar mass of the compound shown in Formula I.

[0040] The present invention also provides a phosphite compound, the structure of which is shown in Formula I:

[0041]

[0042] Among them, R1, R2, R3, R4, R5, R6, and R7 are each independently hydrogen, halogen, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 alkoxy, phenoxy, nitro, cyano, and amide.

[0043] In another preferred embodiment, the naphthylmethyl group in Formula I is selected from the following groups:

[0044]

[0045] The * represents a connection point.

[0046] In another preferred embodiment, the naphthylmethyl group in Formula I is selected from The * represents a connection point.

[0047] The present invention also provides the use of the above-mentioned phosphite compound (compound shown in formula I) in detecting 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite in a sample by high performance liquid chromatography.

[0048] Compared with the prior art, the present invention has the following advantages:

[0049] 1. The detection method provided by the present invention solves the problem that 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite can currently only be quantitatively detected by NMR method, so that 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite can be conveniently and accurately detected without NMR equipment.

[0050] 2. The present invention utilizes naphthalene methanol and compounds with similar structures to convert unstable 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite in the sample to be detected into a stable phosphite triester that can be detected by an ultraviolet detector, so that the solution to be detected obtained after the sample to be detected is treated can be detected by high performance liquid chromatography, and the solution to be detected can be placed for a long time, has good stability, and has high detection accuracy.

[0051] 3. The chromatographic conditions used in the detection method of the present invention ensure that the chromatographic peak has a good shape and high area integration accuracy.

[0052] 4. In the detection method established by the present invention, the phosphorus reagent derivative reference substance has good linearity in the range of 0.2 mg / mL-0.8 mg / mL.

[0053] 5. The detection method of the present invention can detect the content of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite in the sample simply, quickly, at low cost and accurately by high performance liquid chromatography, and is an efficient detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1HPLC spectrum of dinaphthyl-2-cyanoethyl phosphite (compound of formula I-1) obtained by deriving 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite with naphthalenemethanol.

[0055] Figure 2 NMR spectrum of dinaphthyl-2-cyanoethyl phosphite (compound of formula I-1) obtained by derivatizing 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite with naphthalenemethanol (instrument used: Bruker AVANCE III 300).

[0056] Figure 3 LC-MS spectrum of dinaphthyl-2-cyanoethyl phosphite (compound of formula I-1) obtained by derivatizing 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphodiamidite with naphthalenemethanol (instrument used: Waters ACQUITY UPLC H-Class, equipped with QDa detector).

[0057] Figure 4 It is the HPLC spectrum of the test solution obtained by pre-treatment of the sample in the practical application example 1-1, wherein A is the peak of 4,5-dicyanoimidazole, B is the peak of naphthalenemethanol, and C is the peak of dinaphthyl-2-cyanoethylphosphinothrysate (the compound shown in Formula I-1). DETAILED DESCRIPTION

[0058] After in-depth research, the inventors of the present application found that the phosphite triester derivative obtained by reacting 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite in the sample with naphthalene methanol and its similar structural compounds can be detected by high performance liquid chromatography, and its concentration is determined by external standard method, and then the content of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite in the sample is calculated. The detection method does not require an NMR instrument, and has high detection accuracy and good stability.

[0059] In the description of the present invention, the pretreatment of the sample to be tested, that is, the derivatization of 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite, includes the steps of: mixing the sample to be tested containing 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite with an activator and a compound (alcohol) shown in formula III, so that the 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite shown in formula II in the sample to be tested reacts with the compound shown in formula II in the presence of the activator to generate a compound shown in formula I, and the reaction formula is as follows:

[0060]

[0061] Among them, R1, R2, R3, R4, R5, R6, and R7 are each independently hydrogen, halogen, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 alkoxy, phenoxy, nitro, cyano, amide, etc.

[0062] The activator includes but is not limited to 4,5-dicyanoimidazole.

[0063] The molar ratio of the compound represented by formula III to the compound represented by formula II is a conventional amount used in such reactions, for example, preferably 2 to 3:1, more preferably 2.5:1. The molar ratio of the activator to the compound represented by formula II is a conventional amount used in such reactions, for example, preferably 1 to 3:1, more preferably 1.5 to 2.5:1, and further more preferably 1.5 to 2.0:1.

[0064] The reaction temperature is 4 to 40°C, and the reaction time is 2 to 10 minutes. For the preparation of a small amount of the sample to be tested, the activator and alcohol are added to the sample to be tested, shaken for 1 to 3 minutes, and then placed for use. During this process, the 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite is basically reacted.

[0065] During the reaction, no other solvent needs to be added, and the solvent in the sample to be tested can provide the reaction medium.

[0066] During the pretreatment process, the reaction of derivatizing 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite in the sample to be tested to form the compound shown in Formula I can usually be completed within 1 to 10 minutes at room temperature, that is, the derivatization of 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite can be completed during the preparation process. The solvents used in the sample to be tested do not affect the derivatization reaction of 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite.

[0067] In the detection method of 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite of the present invention, the compound represented by formula I used as a reference substance can be obtained by reacting 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite with naphthalenemethanol and a compound with a similar structure thereof according to the method of the present invention.

[0068] The present invention derives 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite into a compound shown in formula I. The solution of the compound shown in formula I is detected by high performance liquid chromatography. When the concentration is 0.2 to 1.5 mg / mL, especially in the range of 0.2 to 0.8 mg / mL, the area peak shown in the liquid chromatogram and the concentration have a good linear relationship. Therefore, the concentration of the compound shown in formula I in the solution to be tested can be accurately determined by the external standard method, and then the content of 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite in the sample to be tested can be calculated.

[0069] In the test solution obtained by pretreatment, the concentration of the compound represented by Formula I obtained by the derivatization reaction is relatively high. To ensure the accuracy of the detection, it is diluted before injection, and the concentration of the diluted test solution is preferably within the linear range of the drawn standard curve. For example, the test solution is diluted 15 to 25 times to a concentration of 0.2 to 1.5 mg / mL, more preferably, 0.3 to 1.2 mg / mL.

[0070] In the description of the present invention, normal temperature refers to 4-40°C, more preferably 15-35°C, and most preferably 20-30°C.

[0071] The present invention will be further described in detail below in conjunction with specific implementation, and the protection content of the present invention is not limited to the following embodiments. Without departing from the spirit and scope of the inventive concept, changes and modifications that can be thought of by those skilled in the art are included in the present invention, and are subject to the protection scope of the attached claims. The process, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art, and the present invention has no special restrictions.

[0072] Example 1

[0073] Preparation of reference substance (compound of formula I-1)

[0074] The preparation method of the reference substance (compound of formula I-1) used to prepare the standard solution in this embodiment comprises the following steps: under nitrogen protection, 1 molar amount of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite is added to a three-necked flask containing 3.0V of toluene, the system is cooled to 5±5°C, 2 molar amounts of 4,5-dicyanoimidazole are added to the system, and then 2 molar amounts of naphthalenemethanol are slowly added dropwise to the system, the temperature is controlled not higher than 10°C, and the temperature is kept warm for 0.5 hour after the addition is completed, the system is warmed to 20±5°C, and stirred for 3 hours. After detecting that the remaining naphthalene methanol is less than 2.0%, 0.5V TEA, 0.3V purified water, and 2.0V n-heptane: toluene = 1:1 are added to the system, and the liquid is allowed to stand and separate. The upper organic phase is washed twice with 0.75% sodium sulfite aqueous solution: N,N-dimethylformamide = 1:1, each time at 5.0V, and the upper organic phase is washed twice with 10% sodium chloride aqueous solution, each time at 5.0V. The organic phase is dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain the product as a light yellow oil. 31 P NMR (400MHz, DMSO-6d) δppm 140.28 (s, 1P); 1H NMR (400MHz, DMSO-6d) δppm 7.90-8.04 (m, 7H), 7.41-7.89 (m, 7H), 5.34 (dJ=7.80, 4H), 4.04 (q, J=8.04Hz, 2H), 2.86 (t, J=5.70Hz, 2H). The prepared reference substance was tested, and the HPLC of the phosphite shown in I-1 was as follows: Figure 1 As shown, its HPLC purity is 83.26%, and its NMR spectrum is as shown Figure 2 As shown, the LC-MS spectrum is as Figure 3 shown.

[0075]

[0076] The HPLC conditions of this embodiment are: Agilent 1260 high performance liquid chromatograph, equipped with UV detector, quaternary pump and automatic sampler; chromatographic column is Waters XBridge C18 (4.6×250 mm, 5 μm); mobile phase: 10 mM ammonium acetate (A)-acetonitrile (B); flow rate: 1 mL / min; analysis time: 35 min; column temperature: 25° C.; detection wavelength: 280 nm; injection volume: 5 μL; gradient elution (0-3 min, 10% B; 3-10 min, 10% B→50% B; 10-20 min, 50% B→90% B; 20-28 min, 90% B; 28-35 min, 10% B).

[0077] Prepare standard solution, draw standard curve, and examine linear relationship

[0078] 100 mg of the reference substance of the compound of formula I-1 was accurately weighed into a volumetric flask, diluted with acetonitrile to make a 5 mg / mL stock solution, and then the stock solution was diluted to 0.25 mg / mL, 0.5 mg / mL, 0.65 mg / mL, 1.0 mg / mL and 1.2 mg / mL standard solutions. The standard solution was injected under the chromatographic conditions of Example 1, and the concentration was used as the abscissa and the peak area was used as the ordinate to perform linear regression. The linear regression equation was obtained as Y=8786240X+95294. The results of the linear relationship investigation are shown in Table 1, and the results show that the linear relationship is good within the concentration range of 0.3-1.2 mg / mL.

[0079] Table 1

[0080]

[0081] Pre-treatment of the samples to be tested:

[0082] A certain amount of 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite solution sample to be tested is measured into a volumetric flask, and 4,5-dicyanoimidazole and naphthalenemethanol are added in an amount of about 2 times the molar amount of 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite, and about 2 times the molar amount of naphthalenemethanol are added, and the solution to be tested is prepared by diluting with acetonitrile, shaking for 1 to 2 minutes, and standing. The solution to be tested contains a compound of formula I-1 generated by the reaction of 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite and naphthalenemethanol, and the reaction formula is as follows:

[0083]

[0084] Detect the concentration of the compound represented by formula I-1 in the test solution

[0085] After the above test solution is diluted 10 to 50 times (for example, the concentration of the diluted test solution is about 0.3 to 1.0 mg / mL), 5 to 20 uL is taken after filtration, and injected under the above chromatographic conditions to obtain a chromatogram. According to the above linear regression equation, the concentration of the compound represented by formula I-1 in the test solution is calculated using the retention time and peak area.

[0086] Calculate the content of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite in the sample to be tested

[0087] The content C of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite in the sample to be tested is calculated using the following formula 1: 样 :

[0088]

[0089] Where: C 样 is the mass percentage content of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite in the sample to be tested, %; C I is the concentration of the compound of formula I in the test solution,

[0090] V is the dilution factor of the test solution,

[0091] m 样 is the weight of the sample to be tested,

[0092] 301.41 is the molar mass of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite,

[0093] 415.43 is the molar mass of the compound represented by formula I-1.

[0094] Practical application example 1

[0095] Pre-treat the sample to be tested: weigh about 150 mg of 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite solution sample 1 into a volumetric flask, wherein the molar amount of 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite contained in the sample 1 is about 0.5 mol, add 1 mol of 4,5-dicyanoimidazole and 1 mol of naphthalene methanol, dilute to volume with acetonitrile, shake well, and use the solution to be tested;

[0096] The above test solution was diluted 20 times (the sample concentration after dilution was about 0.75 mg / mL), 1 mL was filtered, and then injected under the above chromatographic conditions to obtain the chromatogram as shown below: Figure 4 The peak time of Formula I-1 is 22.629 min, and the peak area is 7662010. According to the linear regression equation above, the concentration of the compound represented by Formula I-1 in the test solution is calculated by using the peak area to be 0.86 mg / mL.

[0097] According to the above formula 2, the content of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite in the sample to be tested is calculated to be 81.7%.

[0098] The sample 1 was tested by using an NMR instrument (manufacturer: Bruker, model: AVANCE III 300), and the content of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite was 81.2%, indicating that the test results of the method of the present invention and the NMR test results are very close.

[0099] Precision study of the detection method of Example 1

[0100] Accurately weigh 50 mg of the reference substance of the compound of formula I-1 into a volumetric flask, dilute with acetonitrile to make a 0.5 mg / mL standard solution, and continuously inject the standard solution 6 times under the chromatographic conditions of Example 1, and record the peak area. The results are shown in Table 2. The RSD of the peak area of ​​the reference solution for 6 times is 0.40%, indicating that the detection method of the present invention has good precision.

[0101] Table 2

[0102]

[0103] Repeatability experiment of the detection method of Example 1

[0104] Weigh about 150mg (about 0.5mol) of homemade 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite solution into a volumetric flask, add 1mol4,5-dicyanoimidazole and 1mol naphthalene methanol respectively, dilute with acetonitrile to make a 15mg / mL test solution. Dilute the above test solution 20 times (the sample concentration after dilution is 0.75mg / mL), take 1mL and filter, inject 5uL under the above chromatographic conditions, and obtain the chromatogram. According to the above linear regression equation, the peak area is used to obtain the concentration of the compound shown in formula I-1 in the test solution, and then the content of 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite is calculated using formula 2.

[0105] The above process was repeated 6 times, and the results are shown in Table 3.

[0106] Table 3

[0107] Sample No. The content of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite was measured 1st time 81.7％ 2nd 82.3％ 3rd 81.0％ 4th 81.3％ 5th 82.1％ 6th 80.9％ average value 81.6％ RSD 0.7％

[0108] As can be seen from Table 3, the detection method of the present invention has good repeatability.

[0109] Investigation on the stability of the detection results of the detection method of Example 1

[0110] Weigh about 150 mg (about 0.5 mol) of homemade 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite solution into a volumetric flask, add 1 mol 4,5-dicyanoimidazole and 1 mol naphthalene methanol, dilute with acetonitrile to make a 15 mg / mL test solution. Dilute the test solution 20 times (the sample concentration after dilution is 0.75 mg / mL), inject (filter and inject) at 0, 2, 4, and 8 hours, record the peak area, and the stability test results of the derivative product solution obtained after 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphorodiamidite derivatization are shown in Table 4.

[0111] Table 4

[0112]

[0113] From the results in Table 4, it can be seen that the peak area of ​​the derivative solution at zero time is not much different from the peak area measured at 8 hours, the average value of 4 times is 7649333, and the RSD is 0.7%, indicating that the derivative solution has good stability within 8 hours. It can be seen that the pre-column derivatization according to the method described in this embodiment can be used to simply, quickly and accurately determine the content of the phosphorus reagent.

[0114] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention should be within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A method for detecting 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite, characterized in that: The detection method comprises the following steps: (1) pre-treating the sample to be tested to obtain a test solution, wherein the pre-treatment causes 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite in the sample to be tested to be derivatized into a compound shown in Formula I, (2) using high performance liquid chromatography to detect the derivative product of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite in the test solution, and obtaining the concentration of the derivative product in the test solution by an external standard method, (3) Calculate the content of 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite in the sample to be tested. The pretreatment of the sample to be tested in step (1) includes the steps of: mixing the sample to be tested with an activator and a compound shown in formula III, so that the 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite shown in formula II in the sample to be tested reacts with the compound shown in formula III in the presence of the activator to generate a compound shown in formula I, and the reaction formula is as follows: The naphthylmethyl group in formula I and formula III is selected from the following groups: One of the following, where * represents a connection point, The activator is selected from 4,5-dicyanoimidazole, In the high performance liquid chromatography detection, the chromatographic column used is a C8 column or a C18 column; the mobile phase is composed of an ammonium acetate solution and acetonitrile, an ammonium acetate aqueous solution with a concentration of 5mmol / L to 50mmol / L is used as mobile phase A, acetonitrile is used as mobile phase B, and the gradient elution program is: 0 to 3min, 10% B; 3 to 10min, 10% B→50% B; 10 to 20min, 50% B→90% B; 20 to 28min, 90% B.

2. The method for detecting 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite according to claim 1, characterized in that: The naphthylmethyl group in formula I and formula III is selected from 3. The method for detecting 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite according to claim 1 or 2, characterized in that: In the reaction system, the molar ratio of the compound represented by formula III to the 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite represented by formula II is 2 to 3:1, and / or In the reaction system, the molar ratio of the activator to 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite is 1 to 3:1, and / or The reaction temperature of the reaction system is 4 to 40°C.

4. The method for detecting 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite according to claim 1 or 2, characterized in that: In the high performance liquid chromatography detection, the detection wavelength used is 280nm±4nm.

5. The method for detecting 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite according to claim 1 or 2, characterized in that: The flow rate of the mobile phase is 0.2-2.0 mL / min, and / or The injection volume is 5~20uL.

6. The method for detecting 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite according to claim 1 or 2, characterized in that: The detection method comprises preparing a standard solution containing the compound shown in formula I before using high performance liquid chromatography for detection. The preparation method of the standard solution is: (1) Preparation of linear stock solution: accurately weigh the compound of formula I into a volumetric flask, dilute to volume with acetonitrile to prepare a 6.0-9.0 mg / mL stock solution; (2) Dilute the stock solution into 5 to 8 standard solutions with concentrations ranging from 0.2 to 1.5 mg / mL.

7. The method for detecting 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite according to claim 1 or 2, characterized in that: In step (2), the method for obtaining the concentration of the derivative product in the test solution by the external standard method comprises the following steps: (a) injecting a standard solution containing the compound of formula I into a liquid chromatograph to obtain a liquid chromatogram of the standard solution, performing a linear regression with concentration as the abscissa and peak area as the ordinate to obtain a linear regression equation; and (b) diluting the test solution obtained by the pretreatment and injecting it into a liquid chromatograph, obtaining a liquid chromatogram of the test solution under the same chromatographic conditions as in step (a), qualitatively analyzing the compound of formula I therein by retention time, and calculating the concentration of the compound of formula I in the test solution by peak area according to the linear regression equation.

8. The method for detecting 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite according to claim 7, characterized in that: In step (b), the test solution is diluted 10 to 50 times, and the concentration of the compound of formula I in the diluted solution is 0.3 to 1.2 mg / mL.

9. Use of a phosphite compound in detecting 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite in a sample by high performance liquid chromatography, wherein the phosphite compound has a structure as shown in Formula I: The compound shown in formula I is prepared by mixing a sample containing 2-cyanoethyl-N,N,N′,N'-tetraisopropyl phosphorodiamidite with an activator and a compound shown in formula III, so that the 2-cyanoethyl-N,N,N′,N'-tetraisopropyl phosphorodiamidite shown in formula II in the sample to be tested reacts with the compound shown in formula III in the presence of the activator to generate the compound shown in formula I. The reaction formula is as follows: The naphthylmethyl group in formula I and formula III is selected from the following groups: One of the following, where * represents a connection point, The activator is selected from 4,5-dicyanoimidazole.

10. The use according to claim 9, characterized in that The naphthylmethyl group in formula I and formula III is selected from

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