A novel azide-derived reagent for chiral analysis, its preparation and application
By developing the azide-derived reagent ((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropyl 4-azidobenzoate, the universality and efficiency of chiral analysis of alkynyl compounds were solved, and rapid and accurate chiral separation and quantification were achieved.
Patent Information
- Application Number
- CN202311197187.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-15
- Publication Date
- 2025-07-25
- Estimated Expiration
- 2043-09-15
AI Technical Summary
The existing chiral analysis technology has limited application in complex systems, especially the lack of universality and efficient chiral reagents for the analysis of alkynyl compounds, making it difficult to achieve rapid and accurate separation and quantification.
A azide derivative reagent ((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropyl 4-azidobenzoate was developed to achieve efficient diastereoisomerization and ion mobility mass spectrometry separation by reacting with alkynyl compounds and combining click chemistry.
This azide-derived reagent has high reactivity and wide applicability to alkynyl compounds, and can quickly and accurately perform chiral analysis, simplifying the separation and quantification of alkynyl compounds and improving the analysis efficiency.
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Figure CN117342974B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of testing, and particularly relates to a novel azide derivative reagent for chiral analysis, its preparation and application. Background Art
[0002] Chiral compounds widely exist in nature. Many drug molecules and bioactive molecules such as amino acids and sugars are chiral. Various analytical techniques such as capillary electrophoresis and chromatographic techniques have been widely used in chiral analysis. Among them, chiral liquid chromatography-ultraviolet absorption technology has become the main method for the separation and analysis of chiral compounds at present. However, chromatographic analysis generally requires pre-treatment or purification steps for the analyte, and conditions such as the specifications of chiral chromatographic columns and mobile phases also need to be optimized. The application of chiral chromatography technology in complex systems is very limited. In recent years, the development of ion mobility spectrometry has provided convenience for the analysis of chiral compounds in complex systems. Ion mobility spectrometry (IMS) can separate isomers with the same mass-to-charge ratio (m / z) based on the difference in the migration rates of gaseous ions in a unit electric field within milliseconds. Since the mobility is a function of the ion collision cross section (CCS), and the ion collision cross section depends on the size, shape and charge of the ions in the gas phase, isomers with the same m / z but different CCS can be separated by IMS.
[0003] In 2019, Perez-Miguez et al. (Perez-Miguez, R. et al. Chiral discrimination of DL-amino acids by trapped ion mobility spectrometry after derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate. Anal. Chem. 91, 3277-3285 (2019)) first used (+)-1-(9-fluorenyl)ethyl chloroformate as a chiral reagent to carry out a derivatization reaction with chiral amino acids, and achieved the DL discrimination of common chiral amino acids through ion mobility. At present, chiral analysis techniques of ion mobility spectrometry based on chemical derivatization strategies and ion complexation strategies have been widely applied in fields such as medicine, food safety and life sciences. Chiral alkynyl compounds widely exist in the structures of organic molecules and natural products. Due to the unique reactivity between the alkynyl group and azide compounds, alkynyl compounds have been widely applied in fields such as pharmaceutical and life science research. Currently, the developed chiral derivatization reagents are all based on reactive groups such as acyl chlorides and active esters for chiral analysis of amino acids. It is very meaningful to develop a chiral reagent with better universality for chiral analysis of alkynyl compounds. Summary of the Invention
[0004] In view of the above deficiencies of the prior art, in the first aspect of the present invention, an azide derivative reagent suitable for chiral analysis of alkynyl compounds is provided. The azide derivative reagent is ((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropyl 4-azidobenzoate, and its structural formula is as follows:
[0005]
[0006] In the second aspect of the present invention, a preparation method of the azide derivative reagent of the first aspect of the present invention is provided, including the following steps:
[0007] (1) The carboxyl group in 4-azidobenzoic acid is substituted by chlorine to obtain 4-azidobenzoyl chloride;
[0008] (2) The 4-azidobenzoyl chloride is added to Fmoc-L-phenylalaninol to obtain ((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropyl 4-azidobenzoate, that is, the azide derivative reagent.
[0009] Preferably, the specific method of step (1) is as follows: 4-azidobenzoic acid is dissolved in a solvent to obtain a 4-azidobenzoic acid reaction solution; a catalyst and a chlorine atom donor are sequentially added to the 4-azidobenzoic acid reaction solution and a substitution reaction is carried out, and 4-azidobenzoyl chloride is obtained after the reaction.
[0010] More preferably, the catalyst is N,N-dimethylformamide; the chlorine atom donor is oxalyl chloride or thionyl chloride.
[0011] More preferably, the feeding relationship of each raw material is as follows: the addition amount of 4-azidobenzoic acid is 0.1 - 1000 mmol; the corresponding addition amount of the solvent is 1 - 500 mL; the catalyst is added based on the progress of the induced reaction, and its addition amount is based on observing the generation of bubbles in the 4-azidobenzoic acid reaction solution; the corresponding addition amount of the chlorine atom donor is 1 - 10 equiv.
[0012] More preferably, the substitution reaction is carried out at room temperature or in an ice bath environment, and the reaction duration is 10 min - 12 h.
[0013] Preferably, the specific method of step (2) is as follows: the 4-azidobenzoyl chloride is dissolved in a solvent to obtain a 4-azidobenzoyl chloride solution; then this solution is added to a mixed solution formed by dissolving Fmoc-L-phenylalaninol and an acid-binding agent in a solvent and an addition reaction is carried out, and ((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropyl 4-azidobenzoate, that is, the azide derivative reagent, is obtained after the reaction.
[0014] More preferably, the acid-binding agent includes at least one of triethylamine, tri-n-butylamine, diisopropylethylamine, pyridine, imidazole, 1,8-diazabicycloundec-7-ene, sodium carbonate, sodium bicarbonate, potassium carbonate, and potassium bicarbonate.
[0015] More preferably, the feeding relationship of each raw material is as follows: in the 4-azidobenzoyl chloride solution, the corresponding addition amount of the solvent is 1-500 mL; in the mixed solution, the corresponding addition amount of Fmoc-L-phenylalaninol is 1-10 equiv, the corresponding addition amount of the acid-binding agent is 1-10 equiv, and the corresponding addition amount of the solvent is 1-500 mL.
[0016] Furthermore, the solvent is an aprotic solvent, including at least one of dichloromethane, acetonitrile, tetrahydrofuran, 1,2-dichloroethane, and toluene.
[0017] More preferably, the addition reaction is carried out at room temperature, and the reaction duration is 0.5-72 h.
[0018] In the third aspect of the present invention, there is provided the application of the azide-derived reagent prepared by the method of the first aspect or the second aspect of the present invention, specifically its application as a chiral reagent for chiral analysis of alkynyl compounds.
[0019] Based on the above technical solutions, the synthetic route and inventive concept of the present invention are as follows:
[0020]
[0021] Taking ((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropyl 4-azidobenzoate as the azide chiral derivatizing reagent, for low-polarity alkynyl compounds, the triazole skeleton structure generated by the derivatization reaction has extremely high ionization efficiency during the electrospray ionization process, and has extremely high sensitivity for the analysis of alkynyl compounds in complex systems, enabling rapid chiral analysis of various alkynyl chiral compounds. In addition, while realizing the stereoconfiguration analysis of alkynyl chiral compounds, the present invention can also directly calculate the chiral purity of alkynyl chiral compounds based on the mobility signals of the diastereoisomers generated by the derivatization.
[0022] The design concept of the azide-derived reagent of the present invention is as follows:
[0023] a) The derivatizing reagent needs to contain a single chiral center, preferably transformed from common chiral substances in nature. The chiral structure of this derivatizing reagent is based on the derivative of chiral amino acid, namely Fmoc-phenylpropanol. This raw material is inexpensive, easy to prepare from the corresponding amino acid, and has extremely high optical purity; similarly, the derivatizing reagent can be synthesized based on other various chiral amino acids.
[0024] b) The derivatization reagent should contain a rigid backbone structure to increase the difference in ion mobility of the diastereoisomers generated after derivatization. Through optimization, it is found that structures such as naphthalene ring, adamantane, and fluorenyl have good chiral separation ability.
[0025] c) The derivatization azide reagent is developed for chiral terminal alkynyl compounds and can achieve efficient diastereoisomerization of chiral terminal alkynyl compounds by combining click chemistry.
[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0027] The present invention provides a novel azide derivatization reagent for chiral analysis. This azide derivatization reagent has high reactivity towards alkynyl compounds and good generality.
[0028] The present invention provides a preparation method for the novel azide derivatization reagent for chiral analysis. This method has a simple synthetic route and mild reaction conditions.
[0029] The present invention provides the application of the novel azide derivatization reagent for chiral analysis. When used for chiral analysis of alkynyl compounds with unknown chiral purity, it has the advantages of high efficiency, speed, and high accuracy. Description of the Drawings
[0030] Figure 1 1H NMR spectrum of ((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropyl 4-azidobenzoate synthesized in Example 1;
[0031] Figure 2 13C NMR spectrum of ((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropyl 4-azidobenzoate synthesized in Example 1;
[0032] Figure 3 High-resolution mass spectrum of ((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropyl 4-azidobenzoate synthesized in Example 1;
[0033] Figure 4 Ion mobility spectrum of racemic alkynyl compound 2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)penta-4-ynoic acid (1A) before and after derivatization in Example 2;
[0034] Figure 5 Ion mobility spectrum of racemic alkynyl compound phenyl(4-(prop-2-yn-1-yloxy)phenyl)methanol (2A) before and after derivatization in Example 3;
[0035] Figure 6 Ion mobility plots of the racemized alkynyl compound 2-(2-oxo-2-phenylethyl)hept-6-ynal (3A) before and after derivatization in Example 4;
[0036] Figure 7 Ion mobility mass spectrometry (IMS) analysis results of 2-(2-oxo-2-phenylethyl)hept-6-ynal (3A) with different ee values in Example 4;
[0037] Figure 8 Chiral chromatographic analysis (HPLC) results of 2-(2-oxo-2-phenylethyl)hept-6-ynal (3A) with different ee values in Example 4. Detailed implementation mode
[0038] The present invention will be further illustrated by the following examples, but the present invention is not limited to the scope of the described examples. The experimental methods without specific conditions in the following examples are carried out according to conventional methods and conditions, or selected according to the product specifications.
[0039] In the following examples:
[0040] The analytical instrument used is an ion mobility mass spectrometer, and its model is: electrospray - trapped ion mobility - time - of - flight mass spectrometer Pro (Bruker Daltonics, Germany) timsTOF Pro mass spectrometer (Bruker Daltonics, Germany) with a CaptiveSpray ion source.
[0041] The names and structures of the alkynyl standard compounds used are shown in Table 1:
[0042] Table 1:
[0043]
[0044]
[0045] Example 1
[0046] An azide derivatization reagent is prepared by the following method:
[0047] (1) Add 4-azidobenzoic acid (816 mg, 5 mmol) and 20 mL of anhydrous dichloromethane to a 50 mL single-necked round-bottom flask equipped with a magnetic stir bar. Stir well to completely dissolve 4-azidobenzoic acid to obtain a 4-azidobenzoic acid reaction solution. Add two drops of N,N-dimethylformamide (DMF) to the 4-azidobenzoic acid reaction solution, and then slowly add oxalyl chloride (850 μL, 2 equiv) dropwise to the reaction mixture for a substitution reaction. A large number of bubbles are generated in the reaction solution. After stirring at room temperature or in an ice bath for 30 min, directly concentrate the reaction solution using a rotary evaporator to remove the dichloromethane solvent and excess oxalyl chloride, obtaining a crude product of 4-azidobenzoyl chloride as a white solid;
[0048] (2) Without further purification, dissolve the obtained crude 4-azidobenzoyl chloride in 20 mL of acetonitrile (ACN) to obtain a 4-azidobenzoyl chloride solution. Subsequently, add this solution dropwise to a 50 mL single-necked flask containing Fmoc-L-phenylalaninol (1.87 g, 1 equiv) and pyridine (403 μL, 1 equiv) dissolved in 20 mL of acetonitrile for an addition reaction. After stirring the reaction overnight, the reaction mixture becomes a white suspension and is evaporated to concentrate to remove acetonitrile. The crude product is washed 3 times with ethanol (20 mL) and dried in an oven to obtain a white solid (1.94 g, yield: 75%) of the target product ((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropyl 4-azidobenzoate (denoted as D3), namely the azide-derived reagent.
[0049] As Figures 1 to 3 shown, the obtained product was characterized by 1H NMR, 13C NMR, and high-resolution mass spectrometry in sequence. The structure characterization data of the product are as follows:
[0050] 1 1H NMR (600 MHz, CDCl3) δ 8.01 (d, J = 8.3 Hz, 2H), 7.75 (dd, J = 7.6, 4.0 Hz, 2H), 7.52 (d, J = 7.5 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.35–7.16 (m, 7H), 7.04 (d, J = 8.3 Hz, 2H), 4.95 (d, J = 8.2 Hz, 1H), 4.42–4.26 (m, 4H), 4.17 (t, J = 6.9 Hz, 1H), 2.90–3.15 (m, 2H).
[0051] 1313C NMR (151 MHz, CDCl3) δ 165.76, 155.91, 145.21, 143.92, 141.40, 136.83, 131.65, 129.41, 128.88, 127.83, 127.15, 127.04, 126.27, 125.14, 120.11, 119.04, 66.85, 65.58, 51.53, 47.30, 38.05.
[0052] HRMS (ESI+) exact mass calculated for (C 31 H 26 N4O4) [M + H] + requires m / z 519.2027, found m / z 519.2040.
[0053] The above results indicate that ((S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropyl 4-azidobenzoate was prepared by the method of the present invention. The synthetic route of this method is simple, the reaction conditions are mild, and the yield is relatively high.
[0054] Example 2
[0055] In this example, the racemic 2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)penta-4-ynoic acid (denoted as 1A) was selected as the alkyne standard compound to be analyzed, and was used to test the chiral analysis effect of the azide derivative reagent synthesized in Example 1.
[0056] The racemic 2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)penta-4-ynoic acid was prepared into a 10 mM standard solution with acetonitrile, the azide derivative reagent D3 was prepared into a 10 mM standard solution with dichloromethane, and copper iodide and diisopropyl ethylamine were prepared into a 10 mM standard solution with acetonitrile; 100 μL of each of the above three standard solutions was respectively pipetted into a 1.5 mL centrifuge tube, the reaction solution was ultrasonically reacted at room temperature for 10 min, the copper catalyst was centrifuged and precipitated, and the supernatant was taken and diluted for injection; by setting and optimizing the ion mobility mass spectrometry parameters, the m / z of the derivatized target diastereoisomer was determined, and according to the corresponding [M + Na] + The ion mobility diagram was extracted to judge the diastereoisomer ratio and thus calculate the enantiomeric ratio of the analyte.
[0057] For the comparative experiment, the underivatized 2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)penta-4-ynoic acid (1A) was directly analyzed. As Figure 4 shown, the analysis results indicate that chiral separation cannot be achieved before derivatization, and two mobility peaks with similar peak areas can be separated after derivatization.
[0058] Example 3
[0059] In this example, racemic phenyl(4-(prop-2-yn-1-yloxy)phenyl)methanol (denoted as 2A) was selected as the alkynyl standard compound to be analyzed, for testing the chiral analysis effect of the azide derivative reagent synthesized in Example 1.
[0060] Racemic phenyl(4-(prop-2-yn-1-yloxy)phenyl)methanol was configured into a 10 mM standard solution with acetonitrile, azide derivative reagent D3 was configured into a 10 mM standard solution with dichloromethane, and copper(I) iodide and diisopropylethylamine were configured into a 10 mM standard solution with acetonitrile; 100 μL of each of the above three standard solutions was respectively transferred into a 1.5 mL centrifuge tube, the reaction solution was ultrasonically reacted at room temperature for 10 min, the copper catalyst was centrifuged and precipitated, and the supernatant was taken and diluted for injection; by setting and optimizing the ion mobility mass spectrometry parameters, the m / z of the target diastereoisomer after derivatization was determined, and according to the corresponding [M+Na] + The ion mobility map was extracted to judge the diastereoisomer ratio, thereby calculating the enantiomer ratio of the analyte.
[0061] For the comparative experiment, racemic phenyl(4-(prop-2-yn-1-yloxy)phenyl)methanol (2A) without derivatization was directly analyzed. As Figure 5 shown, the analysis results show that chiral separation could not be achieved before derivatization, and two mobility peaks with close peak areas could be separated after derivatization.
[0062] Example 4
[0063] In this example, racemic 2-(2-oxo-2-phenylethyl)hept-6-ynal (denoted as 3A) and 3A with different enantiomeric excess (ee) values were selected as the alkynyl standard compounds to be analyzed, for testing the chiral analysis effect of the azide derivative reagent synthesized in Example 1.
[0064] 3A and 3A with different ee values were respectively configured into 10 mM standard solutions with acetonitrile, azide derivative reagent D3 was configured into a 10 mM standard solution with dichloromethane, and copper(I) iodide and diisopropylethylamine were configured into a 10 mM standard solution with acetonitrile; 100 μL of each of the above three standard solutions was respectively transferred into a 1.5 mL centrifuge tube, the reaction solution was ultrasonically reacted at room temperature for 10 min, the copper catalyst was centrifuged and precipitated, and the supernatant was taken and diluted for injection; by setting and optimizing the ion mobility mass spectrometry parameters, the m / z of the target diastereoisomer after derivatization was determined, and according to the corresponding [M+Na] + The ion mobility map was extracted to judge the diastereoisomer ratio, thereby calculating the enantiomer ratio of the analyte.
[0065] The comparative experiment directly analyzes the un-derivatized racemized 2-(2-oxo-2-phenylethyl)hept-6-ynal (3A). As Figure 6 shown, the analysis results indicate that chiral separation cannot be achieved before derivatization, and two mobility peaks with similar peak areas can be separated after derivatization. The results of ion mobility spectrometry (IMS) and chiral chromatography analysis (HPLC) are shown in Figure 7 and Figure 8 respectively. By comparing the two figures, it is found that the IMS analysis results are very similar to those of chiral HPLC, indicating the high chiral separation ability and accurate quantification ability of the analysis method and derivatization reagent of the present invention. In addition, after statistics, the separation time of chiral HPLC is about 30 minutes, while the analysis time can be shortened to a few seconds using the azide derivatization reagent D3, greatly improving the analysis speed.
[0066] The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and variations based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by those skilled in the art in the technical field of the present invention through logical analysis, reasoning, or limited experiments based on the concept of the present invention on the basis of the prior art should be within the protection scope determined by the claims.
Claims
1. A chiral analysis azide derivatization reagent applicable to alkynyl compounds, characterized in that, The azide derivative reagent is (S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropyl 4-azidobenzoate, and its structural formula is as follows: 。 2. A method for preparing an azide-derived reagent as described in claim 1, characterized in that, It includes the following steps: (1) The carboxyl group in 4-azidobenzoic acid is substituted by chlorine to obtain 4-azidobenzoyl chloride; (2) The 4-azidobenzoyl chloride reacts with Fmoc-L-phenylalaninol through substitution to obtain (S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropyl 4-azidobenzoate, namely the azide derivative reagent.
3. The method according to claim 2, characterized in that, The specific method of the step (1) is as follows: 4-azidobenzoic acid is dissolved in a solvent to obtain a 4-azidobenzoic acid reaction solution; a catalyst and a chlorine atom donor are sequentially added to the 4-azidobenzoic acid reaction solution and a substitution reaction is carried out, and 4-azidobenzoyl chloride is obtained after the reaction.
4. The method according to claim 3, characterized in that The catalyst is N,N-dimethylformamide; the chlorine atom donor is oxalyl chloride or thionyl chloride; the feeding relationship of each raw material is as follows: the addition amount of 4-azidobenzoic acid is 0.1~1000 mmol; the corresponding addition amount of the solvent is 1~500 mL; based on the progress of the induced reaction, the addition amount of the catalyst is such that bubbles can be observed in the 4-azidobenzoic acid reaction solution; the corresponding addition amount of the chlorine atom donor is 1~10 equiv.
5. The method according to claim 3, wherein: The substitution reaction is carried out at room temperature or in an ice bath environment, and the reaction time is 10 min~12 h.
6. The method according to claim 2, wherein The specific method of the step (2) is as follows: the 4-azidobenzoyl chloride is dissolved in a solvent to obtain a 4-azidobenzoyl chloride solution; then this solution is added to a mixed solution formed by dissolving Fmoc-L-phenylalaninol and an acid-binding agent in a solvent and a substitution reaction is carried out, and (S)-2-(((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-phenylpropyl 4-azidobenzoate, namely the azide derivative reagent, is obtained after the reaction.
7. The method according to claim 6, characterized in that, The acid-binding agent includes at least one of triethylamine, tri-n-butylamine, diisopropylethylamine, pyridine, imidazole, 1,8-diazabicycloundec-7-ene, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate; the feeding relationship of each raw material is as follows: in the 4-azidobenzoyl chloride solution, the corresponding addition amount of the solvent is 1~500 mL; in the mixed solution, the corresponding addition amount of Fmoc-L-phenylalaninol is 1~10 equiv, the corresponding addition amount of the acid-binding agent is 1~10 equiv, and the corresponding addition amount of the solvent is 1~500 mL.
8. The method according to claim 6, wherein: The substitution reaction is carried out at room temperature, and the reaction time is 0.5~72 h.
9. The method according to any one of claims 3 to 8, characterized in that: The solvent uses an aprotic solvent, including at least one of dichloromethane, acetonitrile, tetrahydrofuran, 1,2-dichloroethane, toluene.
10. Use of an azide-derived reagent as described in claim 1 or an azide-derived reagent prepared by the method according to any one of claims 2 to 9, characterized in that: The application of using the azide derivative reagent as a chiral reagent for chiral analysis of alkynyl compounds.