Improved detecting method for solid phase polypeptide synthesis by use of triketohydrindene hydrate

Abstract

The invention provides an improved detecting method for solid phase polypeptide synthesis by use of triketohydrindene hydrate, and the method comprises the following steps of: 1) dissolving 500mg of triketohydrindene hydrate with 12.5ml of boiled distilled water to obtain a yellow solution; adding a vitamin C solution under agitation and continuously agitating after sediment for 15 minutes, moving to a refrigerator to be cooled to 4 DEG C, washing with purified water for 2-3 times, processing with a vacuum drier to obtain reduced triketohydrindene hydrate; 2) weighing 2g of Rink_Amide_MBHA resin, calculating mole number, selecting amino acid with a Fmoc protective radial, reacting for 2 hours under effect of a nitrogen methyl pyrrolidone solution with 8% of carbodiimide to obtain peptide resin, then processing with 25% of a piperidine solution for 30min to obtain peptide resin without the protective radial; and 3) taking 2-5mg of peptide resin without the protective radial, adding 300 microlitres of Buffer A and 75 microlitres of Buffer B, mixing uniformly, adding by water at 100 DEG C for 15min and placing in water at 15-20 DEG C to be cooled to room temperature; and adding 1.2ml of 60% ethanol solution, mixing uniformly, and measuring absorbance and reading the absorbance.

Description

technical field

[0001] The invention relates to the improvement of the ninhydrin detection method, which is to quantitatively measure the free amino groups of polypeptides in solid-phase synthesis. Compared with traditional methods, the determination results directly reflect the degree of condensation and the yield. Background technique

[0002] In the field of peptide synthesis, the judgment of the completeness of the peptide resin synthesis process directly affects the synthesis yield. The conventional method is to directly detect the peptide resin after the condensation is completed, so as to roughly determine the degree of synthesis completion. However, due to some situations in the synthesis that may cause the synthesis reaction to fail to continue, such as: incomplete removal of the protective groups of the resin and amino acids, and protection treatment to avoid the occurrence of wrong sequences after synthesis, etc., if these are used in conventional methods It was j...

Examples

Embodiment

[0023] Implementation steps of the new ninhydrin method:

[0024] (1) Preparation of reaction reagents:

[0025] 1) Preparation of reduced ninhydrin: Weigh 500mg of ninhydrin, dissolve it in 12.5ml of boiling distilled water to obtain a yellow solution, dissolve 500mg of vitamin C in 25ml of warm distilled water, and add it to the ninhydrin solution, and a precipitate appears Then continue to stir for 15 minutes. After completion, move it to the refrigerator to cool to 4°C. After taking it out, the precipitate obtained by filtration is washed with water for 3 times. Store in a vacuum desiccator;

[0026] Buffer A: Mix 5ml pyridine + 1.185g phenol + 5ml ethanol;

[0027] Buffer B: 487.5mg ninhydrin + 12.5mg reduced ninhydrin + 10ml ethanol;

[0028] 25% piperidine solution: mix 25ml piperidine solution + 75ml nitrogen methyl pyrrolidone.

[0029] (2) Sample processing:

[0030] 1) Weigh 2g of Rink_Amide_MBHA resin, and calculate the required resin mole number A according t...

technical field

[0001] The invention relates to the improvement of the ninhydrin detection method, which is to quantitatively measure the free amino groups of polypeptides in solid-phase synthesis. Compared with traditional methods, the determination results directly reflect the degree of condensation and the yield. Background technique

[0002] In the field of peptide synthesis, the judgment of the completeness of the peptide resin synthesis process directly affects the synthesis yield. The conventional method is to directly detect the peptide resin after the condensation is completed, so as to roughly determine the degree of synthesis completion. However, due to some situations in the synthesis that may cause the synthesis reaction to fail to continue, such as: incomplete removal of the protective groups of the resin and amino acids, and protection treatment to avoid the occurrence of wrong sequences after synthesis, etc., if these are used in conventional methods It was j...

Examples

Embodiment

[0023] Implementation steps of the new ninhydrin method:

[0024] (1) Preparation of reaction reagents:

[0025] 1) Preparation of reduced ninhydrin: Weigh 500mg of ninhydrin, dissolve it in 12.5ml of boiling distilled water to obtain a yellow solution, dissolve 500mg of vitamin C in 25ml of warm distilled water, and add it to the ninhydrin solution, and a precipitate appears Then continue to stir for 15 minutes. After completion, move it to the refrigerator to cool to 4°C. After taking it out, the precipitate obtained by filtration is washed with water for 3 times. Store in a vacuum desiccator;

[0026] Buffer A: Mix 5ml pyridine + 1.185g phenol + 5ml ethanol;

[0027] Buffer B: 487.5mg ninhydrin + 12.5mg reduced ninhydrin + 10ml ethanol;

[0028] 25% piperidine solution: mix 25ml piperidine solution + 75ml nitrogen methyl pyrrolidone.

[0029] (2) Sample processing:

[0030] 1) Weigh 2g of Rink_Amide_MBHA resin, and calculate the required resin mole number A according t...

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