Chemical synthesis method of klebsiella toxin tilimycin

Abstract

A chemical synthesis method for the Klebsiella toxin Tilimycin relates to the field of biomedical chemical analysis and synthesis technology. The method includes the following steps: (1) mixing bis(trichloromethyl) carbonate dissolved in a polar aprotic solvent with 3-hydroxy-an-aminobenzoic acid in a certain proportion to undergo a substitution reaction, heating and stirring to obtain intermediate product 1; (2) mixing intermediate product 1 with (S)-2-pyrrolidone dissolved in a polar aprotic solvent in a certain proportion to undergo ammonolysis and hydrolysis reactions, heating and stirring, filtering, and purifying by prep-HPLC to obtain intermediate product 2; (3) mixing intermediate product 2 with pyridine sulfur trioxide dissolved in a polar aprotic solvent and DCM in a certain proportion under alkaline conditions to undergo oxidation and amination reactions, stirring at low temperature, vacuum drying, and purifying by prep-HPLC to obtain the target compound Tilimycin. This invention enables the efficient synthesis of the Klebsiella toxin Tilimycin with high purity and good stability, thus providing a material basis and technical method for scientific research related to this toxin.

Description

Technical Field

[0001] This invention relates to a method for preparing intestinal Klebsiella toxins, specifically to a method for the complete chemical synthesis of Tilimycin. Background Technology

[0002] Tilimycin is a natural product produced by Klebsiella pneumoniae and belongs to the pyrrolobenzodiazepine (PBD) family of heterocyclic compounds. Tilimycin has been confirmed as a cytotoxin that arrests the cell cycle by alkylating DNA, leading to decreased cell stability and ultimately causing cell damage and apoptosis. In antibiotic-associated hemorrhagic colitis (AAHC), the accumulation of tilimycin leads to epithelial cell apoptosis, triggers inflammation, exhibits genotoxicity to host cells, and increases susceptibility to disease in human hosts. Furthermore, studies have reported that tilimycin can promote the mutational evolution of intestinal bacteria, expanding the intestinal resistance group and further exacerbating the public health burden. Given the widespread distribution of Klebsiella pneumoniae in the environment, animals, and humans, this type of toxin presents a broad range of potential applications and has attracted widespread attention from environmental science, medicine, and pharmacy.

[0003] The synthetic pathway of Tilimycin has been identified as being via a non-ribosomal peptide synthase (NPRS) pathway, composed of three proteins: NPSA, THDA, and NpsB. However, the biological synthesis of Tilimycin toxin suffers from poor stability and low efficiency. Furthermore, the lack of standard substances for this toxin further limits in-depth research into its toxicological mechanisms. Therefore, there is an urgent need to develop an in vitro chemical synthesis method for Tilimycin with high yield and purity, good stability and reproducibility. This would contribute to a deeper understanding of Tilimycin's toxicological mechanisms and provide important tools and resources for research in related fields. Summary of the Invention

[0004] The purpose of this invention is to provide a widely applicable, highly efficient, and pure chemical synthesis method for intestinal bacterial toxins. To achieve the above-mentioned objective, this invention provides the following technical solution:

[0005] A synthetic scheme using 3-hydroxy-o-aminobenzoic acid (Formula I) as a substrate, which undergoes cyclization condensation followed by reaction with (S)-2-pyrrolidone to ultimately cyclize and form Tilimycin (Formula IV).

[0006]

[0007] This invention involves using Formula I as a synthetic substrate and reacting Formula I stepwise through three synthetic steps to form Tilimycin (Formula IV).

[0008] (1) The first step reaction scheme of the present invention includes:

[0009] A first reaction mixture comprising compounds of formula A and formula I, dissolved in a moderately polar aprotic solvent, is placed under reaction conditions whereby compounds of formula A and formula I undergo a condensation reaction according to reaction scheme I to form a benzo[a]azine heterocyclic compound of formula II:

[0010]

[0011] Formula A is a halogen compound containing diester and dichloromethyl groups. It is chemically stable and can be used as a protecting group for both NH2 and OH in Formula I.

[0012] Formula I is a common synthetic substrate for the NpsA / ThdA protein peptides that make up Tilimycin. Its amino and carboxyl hydroxyl structures can form amide and ester structures together with Formula A.

[0013] Formula II belongs to benzo[a]a heterocyclic compound and contains a carboxylic acid derivative anhydride structure.

[0014] (2) The second reaction scheme of the present invention includes:

[0015] Under reaction conditions, formula II and formula B, which are dissolved in a highly polar aprotic solvent, undergo hydrolysis and ammonolysis reactions according to reaction scheme II to form a compound of formula III:

[0016]

[0017] Formula B is an organic nitrogen heterocyclic compound used as a chiral catalyst, in which the active hydrogen atom of the nitrogen atom is readily replaced by nucleophilic substitution.

[0018] Formula III is a compound containing an amino group and a five-membered pyrrole nitrogen heterocycle, which is easily alkylated and electrophilically substituted.

[0019] (3) The third reaction step of the present invention includes:

[0020] By placing a compound of formula III and a compound of formula C dissolved in a highly polar aprotic solvent under basic conditions, formula III and formula C undergo electrophilic substitution and amine alkylation reactions according to reaction scheme III to form a compound of formula IV:

[0021]

[0022] In this formula, C is pyridine sulfur trioxide, which can undergo electrophilic substitution at the α-position of the pyrrolidone structure to protect the α-H bond. At the same time, it can further oxidize the hydroxyl groups on the pyrrolidone structure to -CHO, -COOH, and CO2 under the action of strong oxidants.

[0023] Wherein, Formula IV is the compound structure of Tilimycin to be synthesized.

[0024] This invention provides a novel in vitro synthesis method for preparing the intestinal Klebsiella toxin Tilimycin. Compared to traditional microbial synthesis methods, this invention employs simple synthesis steps and readily available starting materials, making the synthesis process simple and easy to operate. Furthermore, this method has low requirements for experimental conditions and equipment, making the synthesis process convenient. Through optimization of the synthesis process and conditions, this method can efficiently synthesize the target product, and the product exhibits good stability and high purity. Therefore, the method of this invention provides a solid material and technical basis for a deeper understanding of the properties and mechanism of action of the intestinal Klebsiella toxin Tilimycin. Attached Figure Description

[0025] Figure 1 The ¹H NMR spectrometry and analysis of the product 8-hydroxy-2H-benzo[d][1,3]azine-2,4(1H)-anhydride prepared in this invention are presented.

[0026] Figure 2 The ¹H NMR spectra and their analysis of the product (S)-(2-amino-3-hydroxybenzene)(2-(hydroxymethyl)pyrrolidone-1-yl) methyl ketone prepared in this invention are presented.

[0027] Figures 3-1 to 3-5 LCMS, HPLC and HNMR of Tilimycin, the product prepared in this invention, and their analysis.

[0028] Figure 4 This is a technology roadmap for the synthesis of the product Tilimycin. Detailed Implementation

[0029] In the first step of reaction scheme I, the reaction conditions include a temperature of about 40°C or higher (however below the melting point of the solute and the boiling point of the solvent in the first reaction mixture), for example, 40°C to 66°C. Preferably, the temperature is about 50°C. More preferably, the temperature is about 45°C.

[0030] In the first step of the reaction scheme I, the compound of formula I is treated with a halocarboxylic acid derivative to form a compound of formula II, wherein formula I, after hydrolysis, forms an anhydride structure together with -NH2 and -OH. Preferably, the halocarboxylic acid derivative is selected from bis(trichloromethyl) carbonate.

[0031] In the first step of reaction scheme I, the first reaction mixture further comprises a polar aprotic solvent. The solvent is selected from: N-methylpyrrolidone, tetrahydrofuran (THF), ethyl acetate (EtOAc), acetone, dimethylformamide (DMF), acetonitrile (MeCN), dimethyl sulfoxide (DMSO), propylene carbonate (PC), chloroform, diethyl ether, and combinations thereof. Preferably, the polar aprotic solvent is selected from THF, chloroform, or diethyl ether. More preferably, the solvent selection takes into account the solubility and solvent toxicity of formula A, choosing a moderately polar aprotic solvent such as tetrahydrofuran.

[0032] In the first step of reaction scheme I, the first reactant further comprises bis(trichloromethyl) carbonate. Preferably, the bis(trichloromethyl) carbonate is present in an amount of about 0.20 molar equivalents (relative to the compound of formula I) to about 0.60 molar equivalents. More preferably, the bis(trichloromethyl) carbonate is present in an amount of about 0.30 molar equivalents to 0.50 molar equivalents.

[0033] In one embodiment of the above-described embodiments, the bis(trichloromethyl) carbonate is present in an amount of about 0.20 molar equivalents to 0.50 molar equivalents. Preferably, the bis(trichloromethyl) carbonate is present in an amount of about 0.35 molar equivalents to about 0.45 molar equivalents. Most preferably, the bis(trichloromethyl) carbonate is present in an amount of about 0.42 molar equivalents. The inventors have found that using bis(trichloromethyl) carbonate in this range facilitates the formation of anhydride bonds in formula II. In this embodiment of the invention, the method may further comprise treating a compound of formula I with bis(trichloromethyl) carbonate, wherein the compound forms a ring structure together with the amino and hydroxyl groups of formula I.

[0034] In step I of the reaction scheme, the yield of synthetic formula II is at least 70% based on the weight of the compound of formula I. Preferably, the yield is at least 75%. More preferably, the yield is at least 80%.

[0035] In the first step of reaction scheme I, the reaction time for compounds of formula A and formula I is 1 to 3 hours. The highest yield of formula II is achieved when the reaction time reaches 3 hours. Preferably, the reaction time is 3 hours.

[0036] In the second step of reaction scheme II, the reaction conditions include a temperature of about 80°C or higher (however below the boiling point of the solvent in the first reaction mixture), for example, 80°C to 110°C. Preferably, the temperature is 90°C or higher. More preferably, the temperature is about 100°C.

[0037] In a preferred embodiment of the second-step reaction scheme II, a five-membered nitrogen heterocyclic structure carrying an active hydrogen is introduced with a compound of formula II to form a compound of formula III, wherein the anhydride bond of formula II acylates the N atom of the five-membered heterocyclic amine. Preferably, the five-membered nitrogen heterocyclic compound is selected from (S)-2-pyrrolidone.

[0038] In a preferred embodiment of the second step reaction scheme II, a five-membered nitrogen heterocyclic structure carrying an active hydrogen is introduced with a compound of formula II to form a compound of formula III, wherein the anhydride bond of formula II acylates the N atom of the five-membered heterocyclic amine. Preferably, the five-membered nitrogen heterocyclic compound is selected from L-proline. Preferably, scheme II may also be selected from:

[0039]

[0040] Formula B-1 is the synthetic substrate of the NpsB protein peptide that makes up Tilimycin, and has an active hydrogen atom on the nitrogen atom.

[0041] Formula III-1 is a compound containing an amino group and a carboxyl group at the α-position of a five-membered pyrrole nitrogen heterocycle, which undergoes a cyclization reaction under certain conditions.

[0042] In the second step of reaction scheme II, the first reaction mixture further comprises a polar aprotic solvent. The solvent is selected from: N-methylpyrrolidone, tetrahydrofuran (THF), ethyl acetate (EtOAc), acetone, dimethylformamide (DMF), acetonitrile (MeCN), dimethyl sulfoxide (DMSO), propylene carbonate (PC), and combinations thereof. Preferably, the polar aprotic solvent is selected from DMSO, DMF, or PC. More preferably, the polar aprotic solvent is DMSO, which has strong oxidizing properties.

[0043] In the second step of reaction scheme II, the first reaction mixture further comprises (S)-2-pyrrolidone. Preferably, the amount of (S)-2-pyrrolidone present is from about 0.80 molar equivalents (relative to the compound of formula II) to about 1.50 molar equivalents. More preferably, the amount of (S)-2-pyrrolidone presents is from about 0.90 molar equivalents to 1.40 molar equivalents.

[0044] In one embodiment of the above-described embodiments, the amount of (S)-2-pyrrolidone present is from about 0.80 molar equivalents to 1.40 molar equivalents. Preferably, the amount of (S)-2-pyrrolidone presents is from about 1.00 molar equivalents to about 1.30 molar equivalents. Most preferably, the amount of (S)-2-pyrrolidone presents is about 1.20 molar equivalents. The inventors have found that using amounts of (S)-2-pyrrolidone within this range facilitates the ammonolysis of the anhydride bond and the acylation of the pentamine in Formula III. In this embodiment of the invention, the method may further comprise treating the compound of Formula II with (S)-2-pyrrolidone, wherein the anhydride bond of Formula II is ammonolyzed.

[0045] In the second step of reaction scheme II, the yield of the synthetic formula II is at least 40% based on the weight of the compound of formula II. Preferably, the yield is at least 50%. More preferably, the yield is at least 55%.

[0046] In the second step, reaction scheme II, the reaction time for compounds of formula B and formula II is 10 to 20 hours. The highest yield of formula III is achieved when the reaction time reaches 16 hours. Preferably, the reaction time is 16 hours.

[0047] In the third step of reaction scheme III, the reaction conditions include approximately 0°C or room temperature (relatively mild reaction conditions). Preferably, the temperature is approximately 0°C.

[0048] In the third step of reaction scheme III, a compound is introduced to protect the α-H bond on the pyrrolidone structure. The compound is selected from: dichloromethane, pyridine sulfur trioxide, and monochloromethane. Preferably, the compound is pyridine sulfur trioxide with pro-oxidant function.

[0049] In the third step of reaction scheme III, the first reaction mixture further comprises a polar aprotic solvent. The solvent is selected from: N-methylpyrrolidone, tetrahydrofuran (THF), ethyl acetate (EtOAc), acetone, dimethylformamide (DMF), acetonitrile (MeCN), dimethyl sulfoxide (DMSO), propylene carbonate (PC), and combinations thereof. Preferably, the polar aprotic solvent is selected from DMSO, DMF, or PC. More preferably, the polar aprotic solvent is DMSO, which has strong oxidizing properties.

[0050] In the third step of reaction scheme III, the first reaction mixture further comprises pyridine sulfur trioxide. Preferably, (S) pyridine sulfur trioxide is present in an amount of about 1.00 molar equivalent (relative to the compound of formula II) to about 3.00 molar equivalent. More preferably, pyridine sulfur trioxide is present in an amount of about 1.50 molar equivalent to 3.00 molar equivalent.

[0051] In one embodiment of the above embodiments, the amount of pyridine sulfur trioxide present is from about 1.50 mol equivalents to 3.00 mol equivalents. Preferably, the amount of pyridine sulfur trioxide present is from about 2.00 mol equivalents to about 3.00 mol equivalents. Most preferably, the amount of pyridine sulfur trioxide present is about 2.50 mol equivalents. The inventors have found that using amounts of pyridine sulfur trioxide within this range facilitates the nucleophilic substitution reaction of the five-membered heterocycle of formula III and the oxidation of the -OH at the α-position. In this embodiment of the invention, the method may further include treating the compound of formula III with pyridine sulfur trioxide, wherein the α-H bond of formula III is protected.

[0052] In the third step of reaction scheme III, the yield of synthetic formula II is at least 30% based on the weight of the compound of formula III. Preferably, the yield is at least 33%.

[0053] In the third step of reaction scheme III, the reaction time for compounds of formula B and formula II is 1 to 3 hours. The highest yield of formula III is achieved when the reaction time reaches 1 hour. Preferably, the reaction time is 1 hour.

[0054] In an embodiment, the method further includes separating the Tilimycin compound from a polar aprotic solvent.

[0055] As used in this invention, unless the context otherwise requires, the term "comprising" and variations thereof, such as "including" and "contained," are not intended to exclude further additives, components, wholes, or steps.

[0056] Further aspects of the invention, as well as those described in the foregoing paragraphs, will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

[0057] An example reaction scheme is as follows:

[0058] Option 1

[0059]

[0060] Option 2

[0061]

[0062] Example:

[0063] Example 1 - Compound of Formula IV

[0064] Example 1A:

[0065]

[0066] L-proline (1.2-1.5 equivalents) was dissolved in DMSO, and then 3-hydroxy-o-aminobenzoic acid (1.00 equivalents) was added to the solution. The entire reaction was carried out under heating and stirring, and the residue was obtained by filtration. The residue was purified by prep-HPLC (Phenomenex Iuna C18 column (250x70mm, 10µm); mobile phase: water (FA)-ethyl acetate; gradient: 0%-30% B; elution time: 20 min) to obtain a brown solid, namely (S)-(2-amino-3-hydroxybenzene)(2-(aldehyde)pyrrolidine-1-yl) methyl ketone (yield 50%-60%).

[0067] Implementing 1B:

[0068]

[0069] A DMSO solution of (S)-(2-amino-3-hydroxybenzene)(2-(aldehyde)pyrrolidine-1-yl)methyl ketone (1.00 equivalent) was stirred at 150-250 °C. The residue was obtained under vacuum and eluted with a Phenomenex Iuna C18 column (250×70 mm, 10 nm) using a gradient elution of water (FA)-ethyl acetate. After 20 min, (11aS)-9,11-dihydroxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one was obtained with a yield of 30-40% and a purity of 95%-98%.

[0070] Example 2 - Compound of Formula IV

[0071] Example 2A:

[0072]

[0073] 16.22 g (0.42 equivalents) of bis(trichloromethyl) carbonate was added to 300 mL of tetrahydrofuran, followed by 20.00 g of 3-hydroxy-o-aminobenzoic acid (1.00 equivalents). The solution was stirred at 45 °C for 3 h, concentrated under reduced pressure, and then 100 mL of n-hexane was added. The precipitate was collected by filtration and dried under vacuum to obtain a grayish-pink powder, which was 22.00 g of 8-hydroxy-2H-benzo[d][1,3]azine-2,4(1H)-anhydride (yield 80.9%). The 1H NMR spectrum of the resulting compound is shown in the appendix to the instruction manual. Figure 1 .

[0074] Example 2B:

[0075]

[0076] 18.29 g of (S)-2-pyrrolidone (1.2 equivalents) was weighed and dissolved in 60 mL of DMSO. Then, 27.00 g of 8-hydroxy-2H-benzo[d][1,3]azine-2,4(1H)-anhydride (1.00 equivalents) was added to the solution. The reaction was stirred at 100 °C for 16 h. The residue was filtered and purified by prep-HPLC (Phenomenex Iuna C18 column (250 x 70 mm, 10 μm); mobile phase: water (FA)-ethyl acetate; gradient: 0%-30% B; elution time: 20 min) to obtain a brown solid, namely (S)-(2-amino-3-hydroxybenzene)(2-(hydroxymethyl)pyrrolidone-1-yl)methyl ketone (20.00 g, yield 56.2%). The 1H NMR spectrum of the generated compound is shown in the appendix to the product manual. Figure 2 .

[0077] Example 2C:

[0078]

[0079] 10.94 g of DIEA (2.50 equivalents) and 13.47 g of SO3·Py (2.50 equivalents) were weighed and dissolved in 32 ml of DCM solution. After mixing with 32 ml of DMSO solution, the mixture was reacted with 8.00 g of (S)-(2-amino-3-hydroxybenzene)(2-(hydroxymethyl)pyrrolidone-1-yl)methyl ketone (1.00 equivalents). The reaction was stirred at 0°C for 1 h. DCM was removed under vacuum to obtain the residue, which was then eluted with a Phenomenex Iuna C18 column (250 × 70 mm, 10 nm) using a water (FA)-ethyl acetate mobile phase for 20 min to yield (11aS)-9,11-dihydroxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one (2.80 g), yield 33.6%, purity 95.2%. The mass spectrometry report of the synthesized compound is attached in image form to the instruction manual. Figures 3-1 to 3-5 .in Figure 3-1 LCMS plot of (11aS)-9,11-dihydroxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one; Figure 3-2 The HPLC chromatogram of (11aS)-9,11-dihydroxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one is shown. Figure 3-3 The HPLC chromatogram of (11aS)-9,11-dihydroxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one is shown. Figure 3-4 The HPLC chromatogram of (11aS)-9,11-dihydroxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one is shown. Figure 3-5 The 1H NMR spectrum of (11aS)-9,11-dihydroxy-1,2,3,10,11,11a-hexahydro-5H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5-one is shown.

Claims

1. A chemical synthesis method for the Klebsiella pneumoniae toxin Tilimycin, characterized in that, Includes the following steps: (1) A substitution reaction was carried out by mixing bis(trichloromethyl) carbonate, which is soluble in a polar aprotic solvent, with 3-hydroxy-o-aminobenzoic acid in a certain proportion, and the mixture was heated and stirred to obtain intermediate product 1 shown in Formula II. ； (2) The obtained intermediate 1 was mixed with (S)-2-pyrrolidone dissolved in a polar aprotic solvent in a certain proportion to undergo ammonolysis and hydrolysis reactions. After heating and stirring, the mixture was filtered and purified by prep-HPLC to obtain intermediate 2 as shown in Formula III. ； (3) The obtained intermediate product 2 was subjected to oxidation and alkylation reactions under alkaline conditions by mixing it with pyridine sulfur trioxide dissolved in a polar aprotic solvent and DCM in a certain proportion. After low-temperature stirring and vacuum drying, the mixture was purified by prep-HPLC to obtain the target compound Tilimycin shown in Formula IV. 。 2. The chemical synthesis method of the Klebsiella toxin Tilimycin according to claim 1, characterized in that, The polar aprotic solvent in step (1) is selected from N-methylpyrrolidone, tetrahydrofuran (THF), ethyl acetate (EtOAc), acetone, dimethylformamide (DMF), acetonitrile (MeCN), dimethyl sulfoxide (DMSO), propylene carbonate (PC), chloroform, diethyl ether, and combinations thereof; the polar aprotic solvents in steps (2) and (3) are selected from N-methylpyrrolidone, tetrahydrofuran (THF), ethyl acetate (EtOAc), acetone, dimethylformamide (DMF), acetonitrile (MeCN), dimethyl sulfoxide (DMSO), propylene carbonate (PC), and combinations thereof.

3. The chemical synthesis method of the Klebsiella toxin Tilimycin according to claim 1, characterized in that, In step (1), the polar aprotic solvent used is tetrahydrofuran, and intermediate product 1 is a benzo[a]azeta-heterocyclic compound; the ratio of bis(trichloromethyl) carbonate and tetrahydrofuran to 3-hydroxy-o-aminobenzoic acid is 16.22g:300ml:20g; the reaction conditions require heating to 45°C and stirring for 3h.

4. The chemical synthesis method of the Klebsiella toxin Tilimycin according to claim 1, characterized in that, In step (2), the polar aprotic solvent used is DMSO, and the intermediate product 2 is a compound containing an amino group and a five-membered pyrrole nitrogen heterocyclic compound. The ratio of (S)-2-pyrrolidone and DMSO to 8-hydroxy-2H-benzo[d][1,3]azine-2,4(1H)-anhydride is 18.69g:60ml:27g. The reaction conditions require heating to 100℃ and stirring for 16h.

5. The chemical synthesis method of the Klebsiella toxin Tilimycin according to claim 1, characterized in that, The prep-HPLC purification liquid chromatography-mass spectrometry conditions in step (2) include a 250x70mm, 10um Phenomenex Iuna C18 column; mobile phase: water FA-ethyl acetate; gradient: 0%-30%B; elution time: 20min.

6. The chemical synthesis method of the Klebsiella toxin Tilimycin according to claim 1, characterized in that, In step (3), the alkaline conditions used to maintain the reaction are DIEA; the polar aprotic solvent used is DMSO; the ratio of pyridine sulfur trioxide, DIEA, DCM, DMSO and the mixture is 13.47g:10.94g:32ml:32ml; the reaction conditions need to start from low temperature 0℃ to room temperature and be stirred for 1h.

7. The chemical synthesis method of the Klebsiella toxin Tilimycin according to claim 1, characterized in that, The prep-HPLC purification liquid chromatography-mass spectrometry conditions in step (3) include a 250×70 mm, 10 nm Phenomenex Iuna C18 column, mobile phase: water FA-ethyl acetate; gradient elution for 20 min.

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