Sulfonate mixture

Abstract

The invention provides a novel method for preparing 1, 10-phenanthroline compound disulfonate, namely an isomer mixture of the 1, 10-phenanthroline compound disulfonate is prepared by sulfonating a 4, 7-diphenyl substituted 1, 10-phenanthroline compound. The obtained isomer mixture has good water solubility, and can be directly used for determination of metal ions in an aqueous solution and related protein quantitative analysis.

Description

Technical Field

[0001] The present invention relates to a new method for preparing 1,10-phenanthroline compound disulfonate. According to the method provided by the present invention, an isomer mixture of 1,10-phenanthroline compound disulfonate suitable for protein quantitative analysis can be obtained;

[0002] 1,10-phenanthroline compounds are widely used in the detection of various metal ions due to their complexing effect on metal ions. For example, 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline (bathocuproine) can be used to determine copper ions in samples; for another example, 4,7-diphenyl-1,10-phenanthroline is often used to determine the concentration of iron ions in samples. Since these compounds have poor water solubility, the technology of introducing hydrophilic groups, such as sulfonate groups, into their structures to increase their water solubility for better application in the detection of related ions in aqueous solutions has been well known. For example, Chinese Patent 201580030382.5 discloses a method for quantifying peptides or proteins by using 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline-3,8-disulfonic acid sodium salt to determine monovalent copper ions in aqueous solution.

[0003] However, the synthesis of pure 1,10-phenanthroline compounds represented by 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline-3,8-disulfonic acid sodium salt (structure H) disclosed in the above patent is very difficult. The lengthy synthesis route and cumbersome column chromatography operation lead to high costs, which seriously restricts its commercial application.

[0004]

[0005] In view of this, the present invention provides a sulfonate mixture, which can be obtained by directly sulfonating commercial 1,10-phenanthroline compounds, is simple to operate, cost-controllable, and easy to industrialize. The obtained sulfonate mixture is highly soluble in water and has excellent response performance to specific metal ions.

[0006] Specifically, the sulfonate mixture provided by the present invention is characterized in that it contains at least two sulfonates, and the at least two sulfonates are selected from two or more of the compounds represented by the following six structures (Structures AF):

[0007]

[0008]

[0009]

[0010] Wherein, R1 and R2 are independently a hydrogen atom, or an alkyl substituent containing 1 to 4 carbon atoms, or a chlorine atom, or a bromine atom, preferably a hydrogen atom and a methyl group;

[0011] M+ is a counter ion of the sulfonate group, preferably a sodium ion, a potassium ion, a lithium ion or an ammonium ion;

[0012] In the sulfonate mixture, at least one sulfonate is derived from a compound represented by structure A, structure B or structure D. Regardless of whether the sulfonate mixture contains one, two or three of the compounds represented by structure A, structure B or structure D, the total mass of the compounds in the sulfonate mixture is not less than 35%.

[0013] A prominent feature of the present invention is that the sulfonate can be prepared from a commercial compound having structure G by a simple one-step sulfonation reaction.

[0014]

[0015] To carry out the sulfonation reaction, the sulfonating agent that can be selected can be one or more of concentrated sulfuric acid, oleum, sulfur trioxide, chlorosulfonic acid and pyridine sulfur trioxide complex;

[0016] When the sulfonation reaction is carried out, the molar ratio of the sulfonating agent to the compound having structure G is at least 2 / 1; under specific reaction conditions, such a ratio ensures that the disulfonate of the compound represented by structure G is obtained, rather than the monosulfonate;

[0017] The sulfonation reaction temperature is -20-180 degrees; the general operation is to mix the compound having structure G and the sulfonating agent at a relatively low temperature in the range of -20 to 20 degrees (preferably -10 to 5 degrees), and then age the sulfonation reaction at a relatively high reaction temperature, generally 20-180 degrees (preferably 60-120 degrees), and the aging time is generally 1-2 hours.

[0018] Under the sulfonation reaction conditions described in the present invention, the sulfonation reaction preferentially occurs on the substituted benzene rings at the 4- and 7-positions of the compound having structure G. No sulfonation reaction at the 3- and 8-positions of the compound represented by structure G was monitored during the reaction, i.e., no generation of the compound having structure H described in Chinese patent 201580030382.5 was found.

[0019] like Figure 1As shown, we mark the reaction sites of the 4-position and 7-position benzene rings of the compound having structure G as p, m, and o, respectively, according to their relationship with the pyridine ring in the phenanthroline matrix. The sulfonation reaction of the present invention has the following characteristics: (1) The two sulfonic acid groups are preferentially distributed on two different benzene rings at the 4-position and the 7-position; that is, the product in which the two sulfonic acid groups are simultaneously distributed on the 4-position benzene ring or simultaneously distributed on the 7-position benzene ring is not detected under the sulfonation conditions of the present invention; (2) The p, m, and o sites of each 4-position or 7-position benzene ring may be sulfonated, but the p site and the m site have higher reactivity.

[0020] BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Compounds with structure G and their reaction sites

[0021] Therefore, based on the characteristics of the sulfonation reaction described in the present invention, the product is a mixture of six sulfonate isomers, namely, pp, pm, po, mm, mo, and oo, which is also the mixture of sulfonates described in the present invention. Although the types and contents of isomers vary according to the specific sulfonation reaction conditions, under the sulfonation reaction conditions described in the present invention, the mixture of sulfonates generated must contain at least one of the compounds represented by structure A (i.e., pp), structure B (i.e., pm), or structure D (i.e., mm).

[0022] The mixture of sulfonates of the present invention has good water solubility, and its solubility in water at 25 degrees Celsius can reach more than 30 grams. In sharp contrast, the compound with structure G is basically insoluble in water, and the compound with structure H described in Chinese patent 201580030382.5 is only 500% more solubility than the compound with structure G. Therefore, the sulfonate mixture of the present invention is particularly suitable for the detection of metal ions in aqueous solution. As mentioned above, 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline (bathocuproin) can be used for the determination of monovalent copper ions in samples; for example, 4,7-diphenyl-1,10-phenanthroline is often used to determine the concentration of iron ions in samples; accordingly, its sulfonation product, that is, the sulfonate mixture of the present invention, can also be used for the determination of corresponding ions.

[0023] A particularly useful application of the sulfonate mixture of the present invention is the detection of monovalent copper ions in aqueous solution. With the aid of a relevant reducing agent, such as ascorbic acid, the sulfonate mixture of the present invention can be used to measure monovalent copper, divalent copper or total copper in a sample, such as an environmental sample or a biological sample.

[0024] According to the application of the sulfonate mixture of the present invention in the determination of monovalent copper ions, another particularly useful use of the sulfonate mixture of the present invention is for quantitative analysis of proteins or peptides in samples. The principle is that proteins or peptides can reduce divalent copper ions to monovalent copper ions; in the presence of the sulfonate mixture, the generated monovalent copper ions and the sulfonate mixture are complexed, and the absorption spectrum is manifested as a new absorption at a maximum absorption wavelength of about 480nm. By measuring the OD value of the absorption generated at about 480nm, the concentration of the protein or peptide in the sample can be correlated.

[0025] The above protein quantitative analysis method is generally carried out in the presence of a buffer, wherein the buffer is an acetic acid-acetate system, a citric acid-citrate system or a tartaric acid-tartrate system, and the concentration thereof is 10-100 mM;

[0026] The divalent copper ions are provided by one or more of copper sulfate, copper nitrate, copper chloride and copper acetate, and the concentration thereof is 0.1-1 mM;

[0027] The total concentration of the sulfonate mixture was 0.01-0.2 mM.

[0028] Features and innovations of the present invention:

[0029] The present invention provides a novel method for preparing sulfonates of 1,10-phenanthroline compounds. According to the technology provided by the present invention, a new type of sulfonates of 1,10-phenanthroline compounds can be conveniently obtained based on commercial raw materials. Such sulfonates have good aqueous solutions and can be used for quantitative analysis of proteins or peptides in aqueous solutions. The technology provided by the present invention is easy to industrialize and solves the key problem of large-scale application of protein or peptide quantitative technology using 1,10-phenanthroline compounds. DETAILED DESCRIPTION

[0030] Example 1: Preparation of 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate disodium salt (chlorosulfonic acid method):

[0031] Add 4kg chlorosulfonic acid to a 5-liter three-necked flask, cool to 0 degrees, add 200g 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline in batches, heat to 120 degrees and react for 2h. Cool, quench the reaction solution with 20 liters of water, filter, and collect the solid. The obtained solid is dissolved in 2 liters of water, adjusted to pH 6-7 with 30% NaOH, and distilled under reduced pressure to remove water. The obtained residue is slurried with 500ml ethanol, filtered, and the solid is collected and dried in vacuo to obtain 300g of 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate disodium salt mixture. HPLC analysis contains three isomers, and the total purity of isomers is 99%

[0032] Example 2: Preparation of 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate disodium salt (oleum method):

[0033] 4kg 20% ​​oleum is added in 5 liters of three-necked flasks, cooled to 0 degree, 200g 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline is added in batches, finished, and warmed up to 70 degree for reaction 1h. Cooling, reaction solution is quenched with 20 liters of water, 7000 grams of sodium chloride are added, stirring and dissolving, cooling and crystallization, and the obtained solid collected by filtration is sulfonic acid. The gained sulfonic acid is redissolved in 2 liters of water, pH 7 is adjusted with 30% sodium hydroxide, and the cooling and crystallization after concentrating and removing part of water are obtained, 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline disulfonate disodium salt mixture 210g is obtained, and HPLC analysis contains four kinds of isomers, and the total purity of isomers is 99%.

[0034] Embodiment 3: Preparation of 4,7-diphenyl-1,10-phenanthroline disulfonate disodium salt 4kg chlorosulfonic acid was added to a 5-liter three-necked flask, cooled to 0 degrees, 200g 4,7-diphenyl-1,10-phenanthroline was added in batches, and the addition was completed, and the temperature was raised to 120 degrees for reaction for 2h. Cooling, the reaction solution was quenched with 20 liters of water, filtered, and solids were collected. The obtained solid was dissolved in 2 liters of water, adjusted to pH 6-7 with 30% NaOH, and water was removed by vacuum distillation. The obtained residue was pulped with 500ml ethanol, filtered, and solids were collected. Vacuum drying gave 230g of 4,7-diphenyl-1,10-phenanthroline disulfonate disodium salt mixture, which contained three isomers by HPLC analysis, and the total purity of isomers was 99%.

[0035] Example 4: Determination of monovalent copper ions in aqueous solution:

[0036] The specific steps include: (1) preparing a concentration of 2*10 -5 , 4*10 -5 , 6*10 -5 , 8*10 -5 , 10*10 -5 (2) preparing a sulfonate mixture with a concentration of 2*10 -4 mol / L, ascorbic acid concentration is 4*10 -4 mol / L solution; (3) Take 20 μL of each solution, mix, incubate for 5 min, and measure the OD value at 480 nm; draw a working curve of OD value versus the concentration of copper sulfate solution, and the curve equation is y=2.07*10 4 x-264.5, linear regression coefficient R=0.9982.

[0037] Example 5: Protein quantitative analysis:

[0038] The specific steps include: (1) accurately weighing 1.13g of a mixture of disodium bathocuproin disulfonate, 0.39g of sodium tartrate and 5.3g of sodium carbonate, dissolving them in water, and fixing the volume to 100ml, which is solution A; (2) weighing 12.5g of copper sulfate pentahydrate, dissolving them in water, and fixing the volume to 100ml, which is solution B; (3) mixing 400 microliters of solution A and 8 microliters of solution B, which is solution C; (4) mixing 20 microliters of solution C and 20 microliters of the sample to be tested, incubating at room temperature (25-30 degrees) for 5 minutes, and measuring the absorption at 480nm with an enzyme marker, and the OD value indicates the protein concentration.

Claims

1. A mixture of sulfonates of 1,10-phenanthroline compounds, characterized in that: It contains at least two of the compounds represented by Structure A, Structure B, Structure C, Structure D, Structure E, and Structure F: Wherein, R1 and R2 are independently a hydrogen atom, or an alkyl substituent containing 1 to 4 carbon atoms, or a chlorine atom, or a bromine atom, preferably a hydrogen atom and a methyl group; M+ is a counter ion of the sulfonate group, preferably a sodium ion, a potassium ion, a lithium ion or an ammonium ion.

2. A mixture of sulfonates according to claim 1, characterized in that The sulfonate mixture contains at least one of the compounds represented by structure A, structure B and structure D, and the total mass of the compounds represented by the three structures accounts for no less than 35% of the sulfonate mixture.

3. The mixture of sulfonates according to claim 1, characterized in that The mixture of sulfonates is prepared by sulfonation reaction using a compound having structure G as a raw material: Wherein, R1 and R2 are independently a hydrogen atom, or an alkyl substituent containing 1 to 4 carbon atoms, or a chlorine atom, or a bromine atom, preferably a hydrogen atom and a methyl group.

4. The sulfonation reaction according to claim 2, characterized in that The sulfonating agent is one or more of concentrated sulfuric acid, fuming sulfuric acid, sulfur trioxide, chlorosulfonic acid and pyridine sulfur trioxide complex.

5. The sulfonation reaction according to claim 2-3, characterized in that: The molar ratio of the sulfonating agent to the compound having structure G is at least 2 / 1.

6. The sulfonation reaction according to claim 2, characterized in that: The temperature of the sulfonation reaction is between -20 and 180 degrees.

7. The sulfonation reaction according to claim 2-4, characterized in that: The sulfonation reaction occurs on the benzene rings at the 4-position and the 7-position of the compound having structure G, and generates at least two of the compounds represented by structures A, B, C, D, E, and F.

8. The mixture of sulfonates according to claim 1, characterized in that The mixture can be used for the determination of metal ions.

9. The mixture of sulfonates according to claim 1 and the determination of metal ions according to claim 7, characterized in that: The mixture of sulfonates can be used for the determination of monovalent copper ions.

10. The sulfonate mixture according to claim 1 and claim 8 is used for the determination of monovalent copper ions, wherein the determination method comprises: (1) mixing an aqueous solution containing the sulfonate mixture with a mixed solution containing monovalent copper ions; and (2) determining the OD value of the absorption spectrum at 480 nm as a signal indicating the concentration of monovalent copper ions.

11. The sulfonate mixture according to claim 1 and the use thereof in determining metal ions according to claims 7-9, characterized in that: The sulfonate mixture can be used for quantitative analysis of proteins or peptides.

12. The quantitative analysis of proteins or peptides according to claim 10, wherein the determination method is: (1) preparing a solution A containing the sulfonate mixture, a buffer and divalent copper ions; (2) mixing solution A with a sample solution containing a protein or peptide; (3) measuring the OD value of the absorption spectrum of the mixed solution at 480 nm as a signal indicating the concentration of the protein or peptide; The buffer is an acetic acid-acetate system, a citric acid-citrate system or a tartaric acid-tartrate system, and its concentration is 10-100 mM; The divalent copper ion is one of copper sulfate, copper nitrate, copper chloride and copper acetate. or several are provided, the concentration of which is 0.1-1 mM; The total concentration of the sulfonate mixture was 0.01-0.2 mM.

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