CN113943243A - Preparation and application of fluorine probe for simultaneously identifying and quantifying amino acid - Google Patents
Preparation and application of fluorine probe for simultaneously identifying and quantifying amino acid Download PDFInfo
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Abstract
Preparation and application of fluorine probe for simultaneously identifying and quantifying amino acid by one-dimensional method19F NMR methods to identify and quantify amino acids in aqueous solutions. The19F NMR methods rely on efficient reactions between fluorine tags and amino acids to produce molecules with distinguishable properties19Stable amide product of F chemical shift. Each derived amino acid has its unique chemical shift and is characteristic19The F chemical shifts and the additional fingerprinting of the derivatized amino acids allow for the identification and quantification of individual amino acids in aqueous solution. The19The F NMR method is proved in biological samples and fetal calf serum, the method established by the invention can be widely used for rapid quantitative analysis of amino acids in metabonomics samples and real biological tissue samples, and has good application prospects in biomedicine and clinical disease diagnosis.
Description
Technical Field
The invention relates to the field of amino acid detection and NMR analysis, in particular to a fluorine probe used for quantitative determination of amino acid and application in biological samples.
Background
Amino acids are not only the basic building blocks of proteins, but are also closely related to many vital activities and play important roles in various biological functions, such as the regulation of important physiological activities of mammalian cells or as neurotransmitters of key energetic processes. Changes in amino acid concentration are indicators of many important physiological activities, such as synaptic transmission, aging, learning, and memory. Since most amino acids do not have strong uv absorption or fluorescence, quantitative analysis of these amino acids usually requires pretreatment or pre-separation. Therefore, the development of an effective method for simultaneously identifying and quantifying amino acids is of great significance in the fields of metabonomics, clinical diagnosis, food science and the like.
Nuclear Magnetic Resonance (NMR) has the advantage of good reproducibility and high fidelity, and a number of free amino acids in metabolites can be determined by specific methods. Traditionally, the application of NMR in the field of metabolomics has relied primarily on one-dimensional techniques1H NMR and natural abundance13C NMR. But for biological samples, the signal may overlap with a complex background. The Raftery group of subjects uses compounds containing enriched isotopes (e.g. containing enriched isotopes)13C and15n) for the chemoselective labeling and analysis of amino-, carboxy-or carbonyl-containing metabolites in complex biological fluids (anal. chem.2010,82, 2303-; chem.2009,81, 4882-. Based on13C and15isotopically labeled N and specific pulse sequences, the free amino acids have been selectively detectable in vivo by NMR. And use of13C and15two-dimensional NMR method using N-isotopically enriched tag for labeling, one-dimensional NMR method using fluorine probe19F NMR analysis is faster and cheaper. In addition, with one dimension1Compared with the H NMR, the method has the advantages that,19f NMR does not require water suppression and has little background signal interference. Thus, the use of fluorine probes shows great advantages for qualitative and quantitative analysis of amino acids. The Fujimoto group performed one-dimensional treatment of amino acids with phthalic acid (OPA) and trifluoro-substituted phenyl mercaptan19F NMR examination, the feasibility of simultaneous detection of amino acids was proposed (Fujimoto, K.anal.chem.2020,92: 1669-1673.). However, the reaction requires a high pH and 50% organic solvent, and the reaction conditions are so severe that it is difficult to quantify the individual amino acids in the analyte and to quantify the amino acids in the biological sample.
Disclosure of Invention
The invention aims to provide a fluorine probe for quantitative determination of amino acid mixtures, determination of amino acid components in biological sample fetal calf serum and quantitative analysis of amino acid content, which aims to overcome the defects of the prior art.
The present invention uses a reactive fluorine probe which is effective to form a stable complex with amino acids in aqueous solution, and each amino acid will be encoded by a feature obtained by reaction with the fluorine probe19F, chemical shift, thereby realizing the detection of the amino acid. In this way, one can directly and simultaneously identify and quantify amino acids in an analyte under mild conditions.
The technical scheme of the invention is as follows:
the present invention relates to a fluorine probe which can react with an amino acid to obtain a stable amide compound19F NMR spectrum shows specific19F, chemical shift.
The fluorine probe has the following structure:
a method for preparing the fluorine probe comprises the following steps: respectively reacting 3-chloro-2-fluorobenzoic acid or 2- (trifluoromethyl) benzoic acid and N-hydroxysuccinimide as raw materials, N, N' -dicyclohexylcarbodiimide as a condensing agent and dichloromethane as a solvent at room temperature for 1-3 hours, filtering after the reaction is finished, concentrating the obtained organic filtrate under a vacuum condition, and finally recrystallizing the obtained crude product with diethyl ether to obtain the probe 1 or the probe 2. Wherein the molar ratio of the 3-chloro-2-fluorobenzoic acid or the 2- (trifluoromethyl) benzoic acid to the N-hydroxysuccinimide to the N, N' -dicyclohexylcarbodiimide is 1:1: 1.
The fluorine probe passes through19Use of F NMR spectroscopy to detect amino acids in aqueous solution. Reacting Probe 1 or Probe 2 with each of the 20 amino acids to obtain the characteristically encoded chemical by19F NMR Spectroscopy for the determination of the substanceIs/are as follows19F chemical shift to achieve qualitative detection of each amino acid.
One such fluorine probe simultaneously detects 20 amino acids. The probe 1 and the probe 2 were reacted with the mixed solution of 20 kinds of amino acids, respectively, and the chemical shifts of the 20 kinds of amino acids in the sample of the mixture were identified by comparing the chemical shifts of the individual amino acids.
One such fluorine probe is reacted with amino acids of different concentrations and a standard curve is drawn according to the peak height of the amide product as a function of concentration, thereby fitting a linear equation.
One of the fluorine probes was used to quantitatively analyze a mixture of 9 essential amino acids in a human body. And calculating the concentration of the amino acid according to a linear equation fitted by the standard curve, and comparing the concentration with the actual concentration so as to realize the quantitative analysis of the amino acid.
The application of the fluorine probe in simultaneous identification and quantitative analysis of amino acids in biological sample fetal calf serum. The fluorine probe acts with fetal calf serum, and qualitative analysis of amino acid in the bovine serum is realized by comparing chemical shift of amino acid marked by the fluorine label. Adding known concentration of amino acid into fetal calf serum sample, treating with excessive fluorine probe, and comparing the concentration of amino acid before and after adding into fetal calf serum19F NMR spectrum peak height difference, thereby realizing quantitative analysis of amino acid in fetal calf serum.
Advantages and advantageous effects of the invention
The synthesis method of the fluorine probe is simple, the reaction of the fluorine probe and the amino acid is carried out in the aqueous solution, and the conditions are mild. The fluorine probe can simultaneously detect and quantitatively analyze amino acid in an aqueous solution and amino acid in biological sample fetal calf serum. The fluorine probe synthesized by the invention has good application prospect in the field of amino acid detection, especially in metabonomics analysis and clinical disease diagnosis.
Drawings
FIG. 1 is a scheme for the synthesis of the fluorine probe of the present invention;
FIG. 2 shows a hydrogen nuclear magnetic resonance spectrum of probe 1 of the present invention;
FIG. 3 is a nuclear magnetic resonance carbon spectrum of probe 1 of the present invention;
FIG. 4 is a hydrogen NMR spectrum of probe 2 of the present invention;
FIG. 5 is a NMR carbon spectrum of probe 2 of the present invention;
FIG. 6 shows the reaction of fluorine probe of the present invention with a single amino acid19F NMR spectrum; wherein a) is the reaction of Probe 1 with a Single amino acid19F NMR spectrum, b) reaction of Probe 2 with a Single amino acid19F NMR spectrum;
FIG. 7 shows the reaction of the fluorine probe of the present invention with a mixture of 20 amino acids19F NMR spectrum; wherein a) is the reaction of probe 1 with a mixture of 20 amino acids19F NMR spectrum; b) for reaction of probe 2 with a mixture of 20 amino acids19F NMR spectrum;
FIG. 8 is a standard curve of the reaction of probe 1 of the present invention with different amino acids; wherein a) is histidine, b) is isoleucine, c) is leucine, d) is lysine, e) is methionine, f) is phenylalanine, g) is threonine, h) is tryptophan, i) is valine;
FIG. 9 is a standard curve of the reaction of probe 2 of the present invention with different amino acids; wherein a) is histidine, b) is isoleucine, c) is leucine, d) is lysine, e) is methionine, f) is phenylalanine, g) is threonine, h) is tryptophan, i) is valine; (ii) a
FIG. 10 is a graph showing simultaneous quantitative determination of the concentrations of nine amino acids in a mixture by the probe 2 of the present invention, compared with the actual concentrations;
FIG. 11 shows the measurement of amino acid content in fetal bovine serum by the fluorine probe of the present invention19F NMR spectrum;
FIG. 12 shows the quantitative determination of amino acid concentration in fetal bovine serum by the fluorine probe of the present invention.
Detailed Description
For a better understanding of the present invention, the technical solutions of the present invention are further described below by means of specific examples and accompanying drawings. However, these examples do not limit the present invention.
Example 1:1, probe 1: 2, 5-dioxopyrrolidin-1-yl 3-chloro-2-fluorobenzoate and probe 2: the synthesis, synthetic route and structural formula of 2, 5-dioxopyrrolidine-1-yl 2- (trifluoromethyl) benzoate are shown in figure 1.
Synthesis of Probe 1: 3-chloro-2-fluorobenzoic acid and N-hydroxysuccinimide are used as raw materials, N, N' -dicyclohexylcarbodiimide is used as a condensing agent, dichloromethane is used as a solvent, the reaction is carried out at room temperature for 2 hours, after the reaction is finished, filtration is carried out, the obtained organic filtrate is concentrated under the vacuum condition, and finally the obtained crude product is recrystallized by using ether, so that the probe 1 is obtained, namely a white solid, wherein the yield is 78.9%.1H NMR(CDCl3,400MHz):7.99-7.96(m,1H),7.74-7.70(m,1H),7.25-7.22(m,1H),2.91(s,1H)ppm.13C NMR(CDCl3,101MHz):168.8,158.6(d,JC-F＝5.1Hz),157.9(d,JC-F＝267.7Hz),136.9,130.7,124.8(d,JC-F＝5.1Hz),123.3(d,JC-F＝17.2Hz),115.4(d,JC-F＝10.1Hz),25.6ppm.19F NMR(CDCl3,376MHz):-107.0ppm.HRMS(ESI)m/z:[M+Na]+calculated for C11H7ClFNO4Na, 293.9940; found,293.9945 nuclear magnetic resonance hydrogen and carbon spectra for probe 1 are shown in fig. 2 and 3. Wherein the molar ratio of the 3-chloro-2-fluorobenzoic acid to the N-hydroxysuccinimide to the N, N' -dicyclohexylcarbodiimide is 1:1: 1.
Synthesis of Probe 2: taking 2- (trifluoromethyl) benzoic acid and N-hydroxysuccinimide as raw materials, N, N' -dicyclohexylcarbodiimide as a condensing agent and dichloromethane as a solvent, reacting at room temperature for 2 hours, filtering after the reaction is finished, concentrating the obtained organic filtrate under a vacuum condition, and finally recrystallizing the obtained crude product with diethyl ether to obtain the probe 2, namely a white solid, wherein the yield is 81.3%.1H NMR(CDCl3,400MHz):8.13(d,J＝4.0Hz,1H),7.86(d,J＝8.0Hz,1H),7.8-7.7(m,2H)2.91(s,1H)ppm.13C NMR(CDCl3,101MHz):168.8,161.0,133.3,132.0,131.4,130.4(q,JC-F＝33.3Hz),127.3(q,JC-F＝5.1Hz),125.4(q,JC-F＝274.7Hz),125.1,25.6ppm.19F NMR(CDCl3,376MHz):-59.7ppm.HRMS(ESI)m/z:[M+Na]+calculated for C12H8F3NO4Na, 310.0298; found,310.0310 nuclear magnetic resonance hydrogen and carbon spectra for probe 2 are shown in fig. 4 and 5. Wherein the molar ratio of the 2- (trifluoromethyl) benzoic acid to the N-hydroxysuccinimide to the N, N' -dicyclohexylcarbodiimide is 1:1: 1.
Example 2: preparation of samples
1) Preparation of Probe 1 and Probe 2 solutions
Probe 1 and probe 2 were prepared in 100mM or 300mM stock solutions, respectively.
2) Preparation of amino acid solutions
20 amino acids, such as glycine (Gly), alanine (Ala), valine (Val), serine (Ser), threonine (Thr), cysteine (Cys), methionine (Met), asparagine (Asn), glutamine (Gln), lysine (Lys), arginine (Arg), histidine (His), proline (Pro), leucine (Leu), isoleucine (Ile), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp) are respectively prepared into 100mM aqueous solution, and the pH is adjusted to 6-9 by NaOH or HCl solution.
3) Preparation of phosphate solutions
50mM phosphate buffer solutions with different pH values (6.5, 7.5 and 8.5) are respectively prepared for amino acid detection and quantitative analysis.
4) Treatment of Fetal Bovine Serum (FBS) samples
Commercial fetal bovine serum samples were filtered with an ultrafiltration tube and the filtrate was used for simultaneous identification and quantitative analysis of amino acids.
Example 3: probe 1 and probe 2 reacted with a single 20 amino acids
20 kinds of amino acids with a final concentration of 0.5mM and a phosphate reaction mixture (pH 7.5) of 3mM probe were prepared, and the reaction mixtures of probe 1 and probe 2 with the amino acids were incubated at room temperature for 3 hours, respectively, and then the reaction was performed19F NMR spectrum collection.
As shown in FIG. 6, the circles indicate the amide products formed by the reaction of the amino acid backbone with the probe, the stars indicate the side chain products, and the diamonds indicate the hydrolysis intermediates of probe 2. In cysteine (Cys), lysine (Lys), lysine (Tyr)And histidine (His), probes 1 (FIG. 6a) and 2 (FIG. 6b), several differences were observed in the reaction mixture19F chemical shift, indicating that the side chain groups of these amino acids also reacted with probe 1 and probe 2. For other amino acids, only one in aqueous solution19F, chemical shift. In summary, the reaction of probe 1 and probe 2 with 20 amino acids, respectively, produces unique and reproducible chemical shifts, as detailed in Table 1 and FIG. 6.
TABLE 1 chemical shifts of backbone amide products from reactions of Probe 1 and Probe 2 with 20 amino acids
Example 4: probe 1 and probe 2 react with 20 amino acids simultaneously
Adding 6mM probe 1 and probe 2 into 20 kinds of amino acid mixture with concentration of 0.1mM, respectively, incubating at room temperature for 3 and 8 hours, and collecting reaction mixture without further treatment19F NMR spectrum. The 20 amino acids in the mixture are simultaneously recognized by probe 1 and probe 2.
As shown in FIG. 7, of the reaction solution19Good separation can be observed by F NMR spectrum19F, chemical shift. For probe 1, the chemical shifts of the amino acids in FIG. 7a match well with the chemical shifts of the individual amino acids shown in FIG. 6a, and only the chemical shifts of leucine (Leu) and arginine (Arg) overlap. Likewise, for probe 2, the chemical shifts of the amino acids in FIG. 7b coincide with the chemical shifts of the individual amino acids shown in FIG. 6 b. The amide products of cysteine (Cys) and aspartic acid (Asp), lysine (Lys) and glutamic acid (Glu), lysine (Lys) and tyrosine (Tyr) overlap, which can be further distinguished by side-chain fingerprinting. But methionine (Met) and asparagine (Asn), arginine (Arg) and alanine (Ala)) The chemical shifts of the main chain amide products of (3) overlap completely (fig. 7 b). Therefore, by combining probe 1 and probe 2, 20 amino acids in the mixture can be simultaneously recognized.
Example 5: drawing standard curves of essential amino acids in 9 human bodies
Nine essential amino acids for human body were selected in this example: valine (Val), leucine (Leu), isoleucine (Ile), threonine (Thr), methionine (Met), phenylalanine (Phe), tryptophan (Trp), lysine (Lys) and histidine (His), standard curves were determined for the quantification of amino acids in real metabolites or tissue samples. Samples having amino acid concentrations of 0.05mM, 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, respectively, were incubated with the excess probe for 5 hours, and then collected19F NMR spectrum. A standard curve was established based on the peak heights of the amide products at different concentrations to fit a linear equation.
As shown in FIGS. 8 and 9, of the amide product19The peak height of the F signal has a correlation with the concentration of the input amino acid, and the probe 1 (FIG. 8) and the probe 2 (FIG. 9) have a good linear relationship, which indicates that the probe 1 and the probe 2 can be well used for quantifying the amino acid in the sample.
Example 6: taking Probe 2 as an example, 9 essential amino acids in the mixture were quantitatively determined
We selected 9 essential amino acids in humans: mixtures of valine (Val), leucine (Leu), isoleucine (Ile), threonine (Thr), methionine (Met), phenylalanine (Phe), tryptophan (Trp), lysine (Lys), and histidine (His) were used as test samples, and two sets of mixtures at concentrations of 0.05mM and 0.1mM, respectively, were prepared for quantitative analysis. The two groups of samples were reacted with the reaction solution of the probe 2 at room temperature for 10 hours, and then collected19F NMR spectra, each set of experiments was repeated three times. The concentration of each amino acid was quantified using a linear equation (FIG. 9) to which an established standard curve was fitted.
As shown in FIG. 10, by comparing the concentration of the amino acids quantified by probe 2 with the actual concentration of the amino acids (gray bar), we can see that there is a good correlation between the concentrations of the 9 essential amino acids determined by probe 2 and the actual concentrations.
Example 7: probe 1 and probe 2 are used for analyzing amino acid components in fetal calf serum
The 5-fold dilution of Fetal Bovine Serum (FBS) filtrate was mixed with 3.0mM Probe 1 and Probe 2, respectively, in 50mM phosphate buffer at pH 7.5 and 8.5, incubated at room temperature for 1h and 4h, respectively, and then recorded19F NMR spectroscopy to identify the composition of amino acids in Fetal Bovine Serum (FBS). Comparison by spectra with probe 1 and probe 2 labeled amino acids (FIG. 7). Up to 13 amino acids are readily recognized in fetal bovine serum, including aspartic acid (Asp), threonine (Thr), serine (Ser), glycine (Gly), phenylalanine (Phe), glutamine (Gln), glutamic acid (Glu), valine (Val), isoleucine (Ile), lysine (Lys), alanine (Ala), leucine (Leu), and arginine (Arg), with triangles representing some species containing active amino groups (fig. 11).
Example 8: probe 1 and probe 2 are used for quantitative analysis of amino acid in fetal bovine serum
To quantify the concentration of amino acids in Fetal Bovine Serum (FBS), we prepared the following samples, respectively: sample 1 is a mixture of 5-fold diluted Fetal Bovine Serum (FBS) filtrate and 3.0mM probe 1 or probe 2; sample 2 was a 5-fold dilution of Fetal Bovine Serum (FBS) filtrate, threonine (Thr), glutamine (Gln), serine (Ser), valine (Val), isoleucine (Ile), glycine (Gly), phenylalanine (Phe), glutamic acid (Glu), and alanine (Ala) each at a concentration of 0.1mM, and a 9.0mM probe 1 mixture. Sample 3 was a mixture of 5-fold diluted Fetal Bovine Serum (FBS) filtrate, and the concentrations of aspartic acid (Asp), threonine (Thr), glutamine (Gln), serine (Ser), valine (Val), isoleucine (Ile) and leucine (Leu) were all 0.1mM and 9.0mM of probe 2. These samples were incubated at room temperature for 2 hours, respectively, and then collected19F NMR spectrum. Samples 2 and 3 were repeated 3 times. By comparing sample 1 with the corresponding amino acids in sample 2 or sample 319The peak height of the F signal determines the concentration of amino acids in Fetal Bovine Serum (FBS).
As shown in FIG. 12, the concentrations of threonine (Thr), glutamine (Gln), serine (Ser), valine (Val), isoleucine (Ile), glycine (Gly), phenylalanine (Phe), glutamic acid (Glu) and alanine (Ala) in the Fetal Bovine Serum (FBS) sample measured by Probe 1 were 0.013, 0.052, 0.017, 0.063, 0.022, 0.057, 0.016, 0.0363 and 0.060mM, respectively. The concentrations of aspartic acid (Asp), threonine (Thr), glutamine (Gln), serine (Ser), valine (Val), isoleucine (Ile) and leucine (Leu) in the Fetal Bovine Serum (FBS) sample determined by probe 2 were 0.005, 0.012, 0.050, 0.018, 0.056, 0.022 and 0.027 mM. Among them, the results of the concentrations of threonine (Thr), glutamine (Gln), serine (Ser), valine (Val) and Ile (isoleucine) measured by probe 1 and probe 2 were consistent. These results indicate that probe 1 and probe 2 can be used to quantify the concentration of amino acids in a biological sample without cumbersome sample handling or pre-separation, which is advantageous and practical compared to other reported methods.
Claims (6)
1. A fluorine probe for simultaneously identifying and quantifying amino acids, the fluorine probe having the formula:
2. a method of preparing the fluorine probe of claim 1, which comprises: 3-chloro-2-fluorobenzoic acid or 2- (trifluoromethyl) benzoic acid and N-hydroxysuccinimide are used as raw materials, N, N' -dicyclohexylcarbodiimide is used as a condensing agent, dichloromethane is used as a solvent, the reaction is carried out for 1 to 3 hours at room temperature, after the reaction is finished, the filtration is carried out, the obtained organic filtrate is concentrated under the vacuum condition, and finally the obtained crude product is recrystallized by ether, thus obtaining the fluorine probe; wherein the molar ratio of the 3-chloro-2-fluorobenzoic acid or the 2- (trifluoromethyl) benzoic acid to the N-hydroxysuccinimide to the N, N' -dicyclohexylcarbodiimide is 1:1: 1.
3. Use of the fluorine probe according to claim 1 for the detection of amino acids in an aqueous solution, wherein: the fluorine probe andthe amino acids being reacted to obtain the signature code19F NMR chemical shift.
4. Use according to claim 3, characterized in that: the fluorine probe can detect 20 amino acids simultaneously.
5. Use according to claim 4, characterized in that: the fluorine probe can quantitatively analyze 9 essential amino acids in a human body.
6. A method of simultaneously identifying and quantifying the amino acids in fetal bovine serum from a biological sample comprising the fluorine probe of claim 1.
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