A phenylboronic acid functionalized covalent organic framework material and applications thereof

Abstract

The application provides a phenylboronic acid functionalized covalent organic framework material and an application thereof. The phenylboronic acid functionalized covalent organic framework material is obtained by taking a COF material containing bromine atoms in a channel as a precursor, 1,4-benzene diboronic acid as a functionalized monomer, and performing functionalized modification on the COF material through a Suzuki-Miyura coupling reaction, and the phenylboronic acid functionalized covalent organic framework material (COF-PBA) has good affinity to cis-dihydroxyl compounds. The material COF-PBA has good adsorption capacity to norepinephrine, adrenaline and dopamine, and a solid-phase extraction column prepared by taking the COF-PBA as an adsorbent is used for pretreatment of human urine, so that enrichment and separation of norepinephrine, adrenaline and dopamine are realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a phenylboronic acid-functionalized covalent organic framework material and its application in the enrichment of catecholamines. Background Technology

[0002] Boric acid affinity materials possess significant advantages such as broad-spectrum selectivity, reversible covalent bonding, pH-controlled capture / release, and rapid adsorption / desorption kinetics, making them important materials for the separation and enrichment of cis-dihydroxy compounds. In the past decade, boron affinity materials have experienced rapid and in-depth development, and boron affinity techniques are considered one of the effective technologies for separating and enriching cis-dihydroxy compounds. Currently, a wide variety of boron affinity materials are known, but the difficulty in functionalizing traditional inorganic porous materials and the poor stability of inorganic-organic hybrid porous materials result in poor adsorption capacity for cis-dihydroxy compounds in these boron affinity materials.

[0003] Covalent organic frameworks (COFs) are an emerging type of porous organic material with advantages such as low density, high crystallinity, large specific surface area, tunable pore size, diverse structures, good acid-base stability, and thermal stability. COFs and their composites have been prepared into highly efficient solid-phase extraction adsorbents and chromatographic stationary phase adsorbents. By introducing highly selective ligands into COFs, the development of porous boron-affinity COFs with abundant boric acid active sites and hydrophilic properties can improve the enrichment selectivity and binding capacity for compounds containing cis-dihydroxy compounds. Furthermore, boric acid-functionalized COFs exhibit high extraction efficiency in the separation and pre-enrichment of trace analytes in complex samples, making them a promising boron-affinity material. Summary of the Invention

[0004] One objective of this invention is to provide a phenylboronic acid-functionalized covalent organic framework material, the structural formula of which is shown below:

[0005]

[0006] In this invention, a COF material containing bromine atoms in its pores (COF-Br) is used as a precursor, and 1,4-phenylboronic acid (PBA) is used as a functionalized monomer. An arylboronic acid in PBA undergoes a cross-coupling reaction (Suzuki–Miyura coupling reaction) with a bromine atom in COF to achieve functionalization modification of COF-Br, resulting in the phenylboronic acid functionalized covalent organic framework (COF-PBA) shown in the above formula. The arylboronic acid in COF-PBA has a good affinity for cis-dihydroxy compounds and can be designed for the enrichment and separation of para-catecholamines (CAs).

[0007] In this invention, the catecholamines (CAs) include norepinephrine (NE), epinephrine (E), and dopamine (DA).

[0008] The synthesis reaction formula for COF-PBA is shown below:

[0009]

[0010] A second objective of this invention is to provide the application of the above-mentioned phenylboronic acid-functionalized covalent organic framework material in the enrichment of catecholamines. Specifically, the application is the enrichment of catecholamines in human urine.

[0011] A third objective of this invention is to provide a solid-phase extraction column using the aforementioned phenylboronic acid-functionalized covalent organic framework material as an adsorbent.

[0012] The fourth objective of this invention is to provide the application of the above-mentioned solid-phase extraction column in the enrichment of catecholamines in human urine.

[0013] The phenylboronic acid functionalized covalent organic framework material (COF-PBA) of the present invention has long-range ordered crystal characteristics and high adsorption capacity for decacatechinamine. Solid phase extraction columns prepared with COF-PBA as adsorbent can be used to enrich norepinephrine, epinephrine and dopamine in human urine. Attached Figure Description

[0014] Figure 1 Infrared spectra of COF-Br and its synthetic raw materials, and COF-PBA.

[0015] Figure 2 The diffraction patterns of COF-Br and COF-PBA powder crystals are shown.

[0016] Figure 3 This is a scanning electron microscope image of COF-PBA.

[0017] Figure 4 TGA curves for COF-Br and COF-PBA.

[0018] Figure 5 The N2 adsorption-desorption isotherm of COF-PBA (the inset shows the pore size distribution of COF-PBA).

[0019] Figure 6 The chromatograms show the liquid phase separation of the components of CAs dissolved in different solvents.

[0020] Figure 7 The effect of eluent volume on elution rate.

[0021] Figure 8 The effect of enrichment solution flow rate on adsorption efficiency.

[0022] Figure 9 The adsorption efficiency of COF-PBA for each component of CAs at different pH values.

[0023] Figure 10 The adsorption capacity of COF-PBA for NE, E, and DA is given.

[0024] Figure 11 Linear fitting equations of the Langmuir model for COF-PBA adsorption of NE(a), E(b), and DA(c).

[0025] Figure 12 HPLC-UV chromatograms of (a) blank urine sample, (b) spiked urine (spiking level: 0.5 μg / mL), (c) urine enriched by COF-PBA extraction column, and (d) eluent.

[0026] Figure 13 The recovery rate of COF-PBA under optimal enrichment conditions corresponds to different enrichment cycles. Detailed Implementation

[0027] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] Example 1

[0031] 1. Synthesis of COF-Br material

[0032] Weigh 21.0 mg of 1,3,5-trialdehyde phloroglucinol (Tp, 0.1 mmol) and 51.3 mg of 3,3'-dibromo-[1,1'-biphenyl]-4,4'-diamine (BD, 0.15 mmol) into a 10 mL pressure-resistant glass tube, then add 1.5 mL of N,N-dimethylacetamide (DMAc), 1.5 mL of 1,2-dichlorobenzene, and 0.2 mL of acetic acid (9 mol / L). Sonicate the mixture for 10 minutes to obtain a homogeneous dispersion. Seal the reaction tube and allow it to react at 120 °C for 72 h. After the reaction, cool the mixture to room temperature, filter, and collect the solid. Wash the solid at least five times consecutively with CH2Cl2, acetone, and THF. Finally, dry the solid under vacuum at 80 °C for 12 h to obtain 40.0 mg of yellow powder COF-Br, with a yield of 80.0%.

[0033] 2. Synthesis of COF-PBA material

[0034] Accurately weigh 20.0 mg COF-Br and 20.0 mg 1,4-phenylenediboric acid (L-PBA) and add them to a 10 mL pressure-resistant glass tube. Then add 0.4 mL of 2 mol / L sodium carbonate aqueous solution and 2 mL of toluene solution. Before using toluene, nitrogen gas should be continuously pumped in for 20 min to remove any oxygen present. Then quickly add 10.0 mg of tetrakis(triphenylphosphine)palladium catalyst. Heat to 80 °C and stir for 48 h under a nitrogen atmosphere. After the reaction is complete, cool to room temperature, collect the precipitate by filtration, wash with deionized water (15 mL × 5), and extract with excess THF. Then wrap the solid powder in filter paper and place it in CH2Cl2 dispersed with mercaptosilica (~1.0 g / 50.0 mg COF-L-Phe) for Soxhlet extraction for 24 h to remove residual Pd catalyst. Finally, dry the solid under vacuum at 80 °C for 24 h to obtain COF-PBA.

[0035] like Figure 1 As shown, comparing the infrared spectra of COF-Br before modification and the functionalized monomer 1,4-phenylenediboronic acid, it can be seen that at 3464 cm⁻¹... -1 ~3050cm -1 A wide range of characteristic absorptions were observed, which was generated by the OH symmetric stretching vibration in the introduced boric acid structure, indicating that the phenylboronic acid structure was successfully modified into the channels of COF.

[0036] like Figure 2 As shown, the PXRD pattern of COF-PBA exhibits a strong diffraction peak on the (100) crystal plane at a small angle of ~3.7° (2θ), indicating that COF-PBA has a good crystal structure. Compared with the PXRD pattern of COF-Br, the PXRD pattern of COF-PBA shows that the diffraction peak at ~5.9° (2θ) disappears, the peak intensity at ~25° (2θ) increases, and a new diffraction peak appears at ~30° (2θ) due to the introduction of the monomer phenylboronic acid. This indicates that the introduction of the phenylboronic acid structure does not destroy the original crystal structure of COF.

[0037] like Figure 3 As shown, the morphological characteristics of COF-PBA were characterized by SEM, revealing a honeycomb-like porous surface on the COF-PBA.

[0038] like Figure 4As shown, thermogravimetric analysis (TGA) was used to evaluate the thermal stability of COF-Br and COF-L-Phe. The partial weight loss of COF-PBA in the 30-100℃ range is due to incomplete removal of free water from the material. In the 100-280℃ range, the weight loss ratio is only about 4.6%, indicating that COF-PBA has good thermal stability. When the temperature rises above 280℃, the weight loss ratio increases significantly, at which point the material structure may be damaged.

[0039] like Figure 5 As shown, COF-PBA exhibits a typical type II reversible isotherm, with a calculated specific surface area of ​​44.51 m². 2 / g.

[0040] Example 2

[0041] 1. Preparation and pretreatment of solid phase extraction columns

[0042] First, COF-PBA is ground, and then 10.0 mg of COF-PBA is weighed and filled into a 1 mL solid-phase extraction column. Both ends of the column are plugged with sieve plates to prevent adsorbent leakage. The column is then washed sequentially with 3 mL of methanol and 3 mL of 0.1 mol / L HCl solution to remove any impurities that may be present in the adsorbent. Next, the column is washed with deionized water, and the pH of the filtrate is measured using pH paper until the pH of the filtrate is neutral. Finally, the column is washed with 3 mL of pH 8.0 phosphate solution. The prepared extraction column is then ready for the adsorption and separation of CAs components. In use, the solid-phase extraction column is connected to a liquid phase pump using an adapter, allowing for precise control of the solution flow rate.

[0043] 2. Optimization of enrichment and elution conditions

[0044] (1) Types of eluents

[0045] To select a suitable eluent, the determination of each component of CAs in different eluents was first investigated. Figure 6 As can be seen, when using 5% formic acid and 5% acetic acid solutions as eluents, the solvents have a certain impact on the chromatographic peaks of the three catecholamine components, making accurate quantification impossible. The impact of 0.01M HCl is minimal, meeting the requirements for accurate quantification of the chromatographic peaks of each CAs component. Therefore, 0.01M HCl was chosen as the eluent in the experiment.

[0046] (2) Effect of eluent volume on recovery rate

[0047] The elution process hydrolyzes the borate ester bonds formed during enrichment, releasing cis-dihydroxy molecules. 0.01M HCl is chosen as the eluent; the amount of eluent used affects the complete dissociation of the formed borate ester bonds. Figure 7 As shown, within the range of 0.5-1.5 mL, the elution rate gradually increases with the increase of the eluent volume. When the amount of eluent reaches 1.5 mL, the elution rate remains stable and reaches over 90%. This indicates that after enriching 10 mL of CAs standard solution in the prepared extractant, 1.5 mL of 0.01 M HCl can basically completely elute the CAs components enriched in COF-PBA.

[0048] (3) Effect of flow velocity on enrichment capacity

[0049] The flow rate determines the interaction time between the target compound and the adsorbent in the solid-phase extraction column, thus affecting the enrichment capacity of COF-PBA for various CAs components. Therefore, this experiment investigated the effect of flow rates in the range of 0.05-2.0 mL / min on the adsorption efficiency. Figure 8 As shown, when the flow rate is less than or equal to 0.2 mL / min, the adsorption efficiency of COF-PBA for the three catecholamines remains around 95%. However, when the flow rate exceeds 0.2 mL / min, the adsorption efficiency of COF-PBA for each CA component gradually decreases. This is because as the flow rate increases, the interaction time between the cis-diol structure in the CA component and the boric acid structure in COF-PBA is too short to form a stable borate ester bond, resulting in a decrease in the enrichment capacity of each CA component on the adsorbent. Therefore, while ensuring good adsorption efficiency, the enrichment time should be minimized as much as possible; in subsequent enrichment experiments, the flow rate was set to 0.2 mL / min.

[0050] (4) Effect of pH on enrichment capacity

[0051] The COF-PBA structure contains abundant boric acid structures. Its affinity for cis-dihydroxy compounds mainly depends on the formation of borate ester bonds under basic conditions. Under acidic conditions, the borate ester bonds hydrolyze to release molecules containing cis-dihydroxy structures. Therefore, pH is one of the important factors affecting the reversible binding process. Higher pH values ​​favor the covalent reaction between boric acid ligands and the ortho-dihydroxy group in cis-dihydroxy compounds, forming five- or six-membered cyclic esters. The effect of pH on enrichment is as follows: Figure 9As shown, within the pH range of 4.0-8.0, the adsorption efficiency of COF-PBA for CAs gradually increases with the increase of sample solution pH. When the solution pH is 8.0, the adsorption efficiency of COF-PBA for each CA component reaches approximately 95%. This indicates that alkaline conditions are more conducive to the formation of stable borate ester bonds between catecholamines and the borate groups in the COF-PBA structure.

[0052] Example 3

[0053] Equilibrium adsorption capacity study

[0054] To investigate the maximum adsorption capacity of COF-PBA for dopamine, adrenaline, and noradrenaline, 10 mL of a mixed standard solution of CAs at concentrations of 5.0, 10, 20, 50, 100, 200, 300, 400, and 500 μg / mL (pH 8.0) was passed through a solid-phase extraction column at a rate of 0.2 mL / min. The filtrate was analyzed by HPLC-UV, and the equilibrium adsorption capacities Q of COF-PBA for NE, E, and DA were calculated. e (μg g -1 The result is as follows Figure 10 As shown.

[0055] Equilibrium adsorption capacity calculation formula:

[0056]

[0057] c0, c e , where V is the concentration (μg / mL) of each component in the stock solution and filtrate, respectively; V is the volume (mL) of the enriched solution; and m is the mass (g) of the adsorbent in the extraction column.

[0058] Langmuir adsorption isotherms were established using equilibrium concentration versus equilibrium adsorption capacity. The Langmuir isotherm model is as follows:

[0059]

[0060] Q max (mg g -1 K is the theoretical maximum adsorption capacity. L (L mg -1 ) is the Langmuir constant, c e Q e These are the concentration of the enriched filtrate (μg / mL) and the equilibrium adsorption capacity (mg / g), respectively. -1 ).

[0061] Depend on Figure 11The maximum adsorption capacities of COF-PBA for norepinephrine, epinephrine, and dopamine were calculated to be 12011 μg / g, 12170 μg / g, and 12300 μg / g, respectively. This indicates that COF-PBA has a good affinity for CAs components.

[0062] Example 4

[0063] Reusability of COF-PBA

[0064] In the boron affinity process, the boric acid structure forms a reversible covalent borate ester bond with the cis-dihydroxy structure. Adsorption and desorption can be completed by adjusting the pH of the system. Therefore, boron affinity materials generally have the advantage of multiple recycling cycles, and the recycling of boron affinity adsorbents can effectively save costs in practical applications. Therefore, the recycling performance of COF-PBA was investigated. The method involved enriching a certain volume of CAs mixed standard solution with COF-PBA, followed by elution. The filtrate and eluent were measured by HPLC, and the elution rate was calculated. The column was then washed with 3 mL of 0.1 M HCl solution, followed by deionized water. The pH of the filtrate was measured using pH paper until it reached neutral. The column was then washed with 3 mL of pH 8.0 phosphate solution to ensure no CAs residue remained in the extraction column. Enrichment and elution were then repeated, and the cycle was repeated to examine the recovery rate of the extraction column after different enrichment cycles. The results are as follows: Figure 13 As shown, after 10 cycles, the recovery rate of each component can still be maintained at around 80%, indicating that the phenylboronic acid-functionalized COF-PBA has good stability and the advantage of multiple cycles.

[0065] Example 5

[0066] To further verify the practical application value of COF-PBA, it was used for the enrichment and analysis of catecholamines in human urine. Since human urine has a weakly acidic pH, the pH needed to be adjusted. Urine from healthy individuals was collected and frozen at -20°C for later use. Before analysis, the urine was thawed at room temperature and then centrifuged at 10,000 rpm for 10 min. The supernatant was adjusted to pH 8.0 and used as the urine sample.

[0067] Take blank urine and add appropriate amounts of CAs standard solution to make the concentrations of the added CAs standard solution 0.5 μg / mL, 1.0 μg / mL, and 1.5 μg / mL, respectively. Then, enrich 30 mL of spiked urine and elute with 1.5 mL of 0.01 M HCl solution. The chromatograms of NE, E, and DA after enrichment and separation with the adsorbent COF-PBA are shown below. Figure 12As can be seen, due to the influence of the urine matrix, the chromatographic peaks of the three catecholamines were weak in urine samples at low spiking levels. However, after enrichment with COF-PBA, the chromatographic peaks of NE, E, and DA were significantly enhanced. The recoveries and relative standard deviations (RSDs) were calculated. The results are shown in Table 1. At different spiking levels, the recoveries ranged from 90.6% to 96.1%, and the RSDs ranged from 1.51% to 3.33%. This indicates that COF-PBA has significant application potential for the enrichment analysis of catecholamines in real-world samples.

[0068] Table 1. Intra-day recovery (R) and RSD (n=9) of urine samples enriched with NE, E, and DA at different spike concentrations.

[0069]

[0070] The results above show that the solid-phase extraction column prepared with COF-PBA as the adsorbent can be used to enrich norepinephrine, epinephrine and dopamine in human urine.

Claims

1. The application of phenylboronic acid-functionalized covalent organic framework materials in the enrichment of catecholamines, characterized in that, The structural formula of the phenylboronic acid-functionalized covalent organic framework material is shown below: ； The catecholamine is norepinephrine, epinephrine, or dopamine.

2. The application of solid-phase extraction columns in the enrichment of catecholamines in human urine, characterized in that, The solid-phase extraction column uses a phenylboronic acid-functionalized covalent organic framework material as the adsorbent, and the structural formula of the phenylboronic acid-functionalized covalent organic framework material is shown below: ； The catecholamine is norepinephrine, epinephrine, or dopamine.

Images