Preparation method of adenosine-2′,3′-cyclic phosphate_Industrial additives

Background and overview^[1]

Cyclic nucleotides are important second messengers in cells and participate in the regulation of multiple signal transduction pathways in cells. The second messengers currently known to be discovered include cyclic adenosine monophosphate (3′,5′-cAMP), cyclic guanosine monophosphate (3′,5′-cGMP), and cyclic cytidine monophosphate (3′,5′ -cCMP), cyclic uridine monophosphate (3′,5′-cUMP), cyclic diguanylate (c-di-GMP), cyclic diadenylate (c-di-AMP) and cyclic guanosine adenosine acid (cGAMP). In addition to the cyclic nucleotide second messengers that have been discovered above, in 2009 Edwin et al. also discovered adenosine-2′,3′-cyclic phosphate (2′) in human kidney cells for the first time. ,3′-cAMP), and subsequently the presence of adenosine-2′,3′-cyclic phosphate was discovered in mammalian brain and other tissue cells. The researchers also detected adenosine-2′,3′-cyclic monophosphate and guanosine-2′,3′-cyclic monophosphate (2′,3′-cGMP) from plant cells. The presence of cytidine-2′,3′-cyclic monophosphate (2′,3′-cCMP) and uridine-2′,3′-cyclic monophosphate (2′,3′-cUMP) was detected in the bacteria. Existing research results indicate that these four 2′,3′-cyclic nucleoside monophosphates are likely to represent another new type of second messenger in biological cells. As 2′,3′-cyclic nucleoside monophosphates attract more and more attention, their demands in scientific research and medicine are also increasing. At present, the preparation of 2′,3′-cyclic nucleoside monophosphate adopts chemical synthesis method, and there is no biological preparation method. Chemical synthesis to produce 2′,3′-cyclic nucleoside monophosphate has low yield and high price, which limits its development and application to a certain extent.

Preparation^[1]

Step 1: Extraction of 2′,3′-cyclic nucleoside monophosphate mixture

(1) Let the eluted target protein LfliZ stand at 0°C for 3 days. At this time, there will be obvious precipitation in the protein solution. Centrifuge the protein solution 1 poloxamer at 188 2000 rpm for 20 minutes to obtain the supernatant;

(2) Ultrafiltrate the supernatant obtained in step (1) with an ultrafiltration tube with a molecular weight cutoff of 3K to remove residual proteins. The ultrafiltration membrane filtrate contains four kinds of 2′, 3 ′-cyclic antioxidant 168-shaped nucleoside monophosphate solution;

Step 2: Pure products of four cyclic nucleotides adenosine-2′,3′-cyclic phosphate, 2′,3′-cGMP, 2′,3′-cCMP and 2′,3′-cUMP Extract

(1) The obtained 2′,3′-cyclic nucleoside monophosphate solution was separated by high performance liquid chromatography using a C18 reverse chromatography column to obtain four pure products of 2′,3′-cyclic nucleoside monophosphate. ;The mobile phase used is: Liquid A, 10mM ammonium acetate solution, pH 5.0; Liquid B, 75% Liquid A plus 25% methanol;

(2) The separation program used in the above steps is: 0min, liquid B 0%; 2.5min, liquid B 0%; 5min, liquid B 30%; 10min, liquid B 60%; 14min, liquid B 100% ;21min, liquid B 100%; 22min, liquid B 50%; 23min, liquid B 0%; 30min, liquid B 0%. The flow rate is 10mL/min, and the detection wavelength is 254nm.

Step 3: Detection of the yield of four pure 2′,3′-cyclic nucleoside monophosphates

(1) Dilute the four purchased 2′,3′-cyclic nucleoside monophosphate standards with distilled water to the following concentration gradient: 0μM, 20μM, 40μM, 60μM, 80μM and 100μM, using a C18 reverse chromatography column. Perform high-performance liquid chromatography to detect the peak area corresponding to the cyclic nucleoside monophosphate standard at each concentration, and then draw a standard curve with the peak area as the abscissa and the concentration as the ordinate.

(2) Using the prepared standard curve, conduct high-performance liquid chromatography on the prepared four 2′,3′-cyclic nucleoside monophosphate samples with a C18 reverse chromatography column to detect the concentration. The culture medium can extract 1.30±0.17μmol of 2′,3′-cCMP, 1.40±0.12μmol of 2′,3′-cUMP, 2.81±0.25μmol of 2′,3′-cGMP and 2.61±0.19μmol of adenosine. -2′,3′-Cyclic phosphate. According to the molecular weights of the four 2′,3′-cyclic nucleoside monophosphates, converting the above molar amounts into masses, it can be obtained that approximately 2.5 mg of the four pure 2′,3′-cyclic nucleosides can be extracted per liter of culture medium Glycoside monophosphate.