15 results about "Hydroxyprogesterone" patented technology

The present invention relates to a method for detecting steroid hormones in serum, comprising the following steps: (1) adding 0.1 to 20 mg of a magnetic solid phase extractant to each milliliter of a sample to be tested, mixing, magnetically separating, and removing liquid; adding a washing solution, mixing, magnetically separating, and removing liquid; adding an eluent for elution, mixing, magnetically separating, and collecting the eluent to obtain a test solution; (2) detecting the hormone content in the test solution by liquid chromatography and tandem mass spectrometry; wherein the hormone is selected from any one of testosterone, androstenedione, dihydrotestosterone, dehydroepiandrosterone sulfate, and 17α-hydroxyprogesterone or a combination thereof; the magnetic solid phase extractant comprises a magnetic core ferrosoferric oxide, a middle layer structure silica wrapping the magnetic core, and an outer layer mesoporous structure, wherein the outer layer mesoporous structure is selected from any one of polystyrene and polystyrene divinylbenzene or a combination thereof. The present invention has the characteristics of good stability, high accuracy and sensitivity, strong specificity, high recovery rate, simple and controllable operation, fast analysis speed, etc., and can be used for simultaneous quantitative detection of multiple hormones in clinical practice.

The invention discloses a preparation method of 17[alpha]-hydroxyprogesterone, wherein the preparation method comprises the steps: refining: step 1, dissolving 1 W of a crude product into a mixed solution of 4-5 V dichloromethane and 6-8 V ethanol, and heating to dissolve the crude product; step 2, weighing activated carbon exceeding a preset amount, then putting the activated carbon into a drying device for drying, controlling the drying temperature to be 60-85 DEG C, and controlling the drying time to be 10-15 minutes; and step 3, taking out the activated carbon dried in the step 2, and then placing the activated carbon in a grinding device for fine grinding. According to the scheme, activated carbon in the refining process in an original scheme is finely processed and then added, so that the reaction rate is effectively increased, the time of reflux decoloration is shortened, large-scale production and processing are facilitated, and the production cost is further reduced.

The invention provides a chemical synthesis method of 20 alpha-hydroxyprogesterone, and belongs to the technical field of organic synthesis. The invention provides a simple and efficient synthesis path, and high-purity 20 alpha-hydroxyprogesterone can be obtained with good yield and extremely high stereoselectivity and specificity. The method is easy and convenient to operate and good in reproducibility, and effectively overcomes the defects that a product is an isomer mixture, separation and purification are difficult, and the target product yield is low in a traditional method. The successful implementation of the invention provides a stable and reliable material source for obtaining sufficient high-purity 20 alpha-hydroxyprogesterone, powerfully supports the subsequent activity research, dosage form development and new drug creation of the compound, and has important significance in the field of health drugs for women, health protection and health protection. The compound shows important application value and potential in the field of research and development of novel progesterone drugs.

The application discloses a steroid C14 alpha hydroxylase CLCYP14 mutant, relates to the field of proteins, and applies a site-directed saturation mutation technology and a combination mutation strategy to screen and obtain CLCYP14 mutants I111L-M115K and I111L-V124W with high hydroxylation specificity. Compared with CLCYP14, the hydroxylation selectivity and conversion rate of I111L-M115K and I111L-V124W for steroid compounds progesterone, 21-hydroxyprogesterone, 5beta-hydroxyprogesterone, 1-dehydroprogesterone, 21-hydroxypregna-1,4-diene-3,20-dione and deoxycortisone are obviously improved.

The invention provides a P450 enzyme CYP5103-BM capable of catalyzing 14 alpha-hydroxylation of progesterone, and a mutant CYP5103-BM-Y291H of the P450 enzyme CYP5103-BM. Wherein the gene sequence of the P450 enzyme CYP5103-BM is as shown in SEQ ID NO: 1, and the gene sequence of the mutant CYP5103-BM-Y291H is as shown in SEQ ID NO: 3. The transformants respectively carrying the CYP5103-BM gene and the CYP5103-BM-Y291H gene can be subjected to a whole-cell biological catalytic reaction to convert a substrate progesterone into 14 alpha-hydroxyprogesterone, and the conversion rates in 36 hours respectively reach 50.04% and 88.76%. The mutant CYP5103-BM-Y291H has higher catalytic activity on progesterone, the production cost of 14 alpha-hydroxyprogesterone can be greatly reduced, the production time can be shortened, and the mutant CYP5103-BM-Y291H has extremely high application value for industrial production of progesterone and research of C14-hydroxylated steroid drugs.

The invention discloses a self-assembly double-enzyme compound, a preparation method and application of the self-assembly double-enzyme compound in synthesis of a prednisone intermediate, and the self-assembly double-enzyme compound is obtained by fusing 11 alpha-hydroxylase and 1, 2-dehydrogenase on the basis of a SpyCatcher / SpyTag system. According to the method, a self-assembled double-enzyme compound of 11alpha-hydroxylase and 1, 2-dehydrogenase is constructed through a SpyCatcher / SpyTag system, 17alpha-hydroxyprogesterone is taken as a substrate, double-enzyme cascade one-step catalytic reaction is realized, the reaction process is simplified, and separation and purification operation of an intermediate product is omitted. The self-assembled double-enzyme compound constructed by the invention forms a high-efficiency substrate channel, accelerates the rapid transfer of an intermediate from a catalytic activity center of a first enzyme to a catalytic activity center of a second enzyme, improves the catalytic efficiency, is suitable for industrial application, and has a conversion rate of 99.6%.

The invention provides a microbial enzyme catalytic synthesis method of 20 alpha-hydroxyprogesterone, and belongs to the technical field of biosynthesis. The invention establishes an efficient, green and high-selectivity 20 alpha-hydroxyprogesterone biosynthesis method, and the method has the direct effects that the directional hydroxylation of the C20 site of the substrate progesterone is realized by utilizing a recombinase technology, the generation of byproducts is effectively avoided, the subsequent separation and purification process is simplified, the reaction conditions are mild, the cost is low, and the method is suitable for industrial production. The energy consumption and the safety risk are reduced; from the aspect of product application significance, the method provides a stable and reliable material source for large-scale preparation of 20 alpha-hydroxyprogesterone, the environmental protection property and economical efficiency of the production process are remarkably improved, a solid material foundation is laid for in-depth study on the biological activity and application of the compound, and the method is suitable for industrial production. And a beneficial technical reference is provided for green biological manufacturing of other steroid hormone medicines.

The purpose is to provide plasma biomarkers for bone infections in humans. [Solution] The plasma biomarker for bone infection consists of one or more substances selected from the group consisting of sphingosine-1-phosphate, taurocholic acid, glycololic acid, 11a,b-hydroxyprogesterone, 15(S)-HETE-2, 13-dehydrochenodeoxycholic acid, 3,21-dihydroxy-5a-pregnan-20-one-2, aldosterone, bisacrone-2, naringenin, tauro-a-muricholic acid-6, 1-deoxysphingosine, 12(S)-HETE-2, 2-arachidonoylglycerol, 3-hydroxydodecanoic acid, 3-hydroxytetradecanoic acid, 3b-hydroxy-5-cholestenoic acid, AEA(22:4)-1, AEA(22:4)-2, bilverdin, sphingosine, malic acid, and succinic acid.

The invention provides a preparation method of 17 [alpha]-hydroxyprogesterone, which comprises the following steps: S1, putting 17 [beta]-cyano-17 [alpha]-hydroxy-4-androstene-3-ketone, ethylene glycol, triethyl orthoformate and a beta-CD-SO3H / SiO2 catalyst into a reaction kettle, and carrying out a ketal reaction, so as to obtain 17 [beta]-cyano-5-androstene-17-ol-3, 3-ethylene ketal; and S2, dissolving the 17 [beta]-cyano-5-androstene-17-ol-3, 3-ethylene ketal in an organic solvent, adding lithium chloride and a benzene solution containing methyl zinc, carrying out a reaction, and after the reaction is finished, carrying out acid hydrolysis to obtain the 17 [alpha]-hydroxyprogesterone. The solid acid catalyst beta-CD-SO3H / SiO2 coated hydrophobic SiO2 is adopted, so that the problems of more side reactions, low purity and yield and the like of liquid acid are effectively solved, and the purity and yield of the final product respectively reach 98.8% and 92.9%.

This invention relates to the field of biotechnology, and more particularly to a P450 enzyme mutant for steroid C11α-hydroxylation, the amino acid sequence of which is shown in SEQ ID NO:2. In a whole-cell catalytic system of *Saccharomyces cerevisiae*, this P450 enzyme mutant achieved a 14-fold increase in the conversion rate of 17α-hydroxyprogesterone compared to the wild type. Expression of this mutant in a systematically modified *Pichia pastoris* chassis strain increased the substrate conversion rate to 96.5% and the product selectivity to 99.1%. Scale-up culture at 2.5 L, with a single feed of 60 g of substrate, yielded a 11α,17α-dihydroxyprogesterone titer of 24.8 g / L. Furthermore, this mutant also exhibited good catalytic activity and C11α-hydroxylation selectivity for various steroid drugs such as progesterone and canrenone, demonstrating a broad substrate spectrum and promising prospects for industrial applications.

The application provides a preparation method of 17alpha-hydroxyprogesterone, which comprises the following steps: S1, 17beta-cyano-17alpha-hydroxy-4-androsten-3-ketone, ethylene glycol, triethyl orthoformate and a beta-CD-SO3H / SiO2 catalyst are put into a reaction kettle to perform a ketal reaction, so that 17beta-cyano-5-androsten-17-ol-3,3-ethylene glycol ketal is obtained; S2, the 17beta-cyano-5-androsten-17-ol-3,3-ethylene glycol ketal is dissolved in an organic solvent, lithium chloride and a methyl zinc-containing benzene solution are added to react, and after the reaction is completed, 17alpha-hydroxyprogesterone is obtained through acid hydrolysis. The solid acid catalyst beta-CD-SO3H / SiO2@hydrophobic SiO2 is adopted, so that the problems of more side reactions, lower purity and lower yield caused by liquid acid are effectively solved, and the purity and the yield of the final product reach 98.8% and 92.9% respectively.

The invention discloses an improved synthesis method of 17α-hydroxy-1,4,6-pregnanetriene-3,20-dione. The steps are: 1) under inert atmosphere protection, 17α-hydroxyprogesterone, acid and dichlorodicyanobenzoquinone react at 0-10 DEG C, wherein dichlorodicyanobenzoquinone is added after the reaction system is cooled to 0-10 DEG C, and the reaction is complete; 2) the reaction solution obtained in step 1) is warmed to 30-40 DEG C, dichlorodicyanobenzoquinone is added, and the temperature is continued to be raised to reflux, and the reflux reaction is carried out until the reaction is complete; 3) post-treatment obtains 17α-hydroxy-1,4,6-pregnanetriene-3,20-dione. The synthetic method has high yield and high purity, and is prepared by a one-pot method, is easy to operate, has high production efficiency, and is green and environmentally friendly.

The application discloses a 21-hydroxylase deficiency screening kit and application thereof, relates to the blood analysis technical field, and a steroid hormone marker composition of 21-hydroxylase deficiency, wherein the nine markers comprise 17alpha-hydroxyprogesterone, androstenedione, 11-deoxycortisol, 21-deoxycortisol, cortisol, corticosterone, progesterone, dihydrotestosterone and 11-deoxycorticosterone; the kit can be used for screening 21-hydroxylase deficiency, 11beta-hydroxylase deficiency, 17alpha-hydroxylase deficiency and other types of diseases; the kit comprises detection components for detecting the hormones; compared with a traditional time-resolved fluorescence immunoassay method, the LC-MS / MS multi-index combined detection scheme adopted in the application can significantly reduce false positive results; and invalid recall, unnecessary family anxiety and medical burden caused by false positive results are greatly reduced.

The present invention discloses a synthesis method of deuterated megestrol acetate and its application. The synthesis method comprises: first, dehydrogenating 17α-hydroxyprogesterone-17-acetate I under the oxidative action of tetrachlorobenzoquinone to obtain a diene compound II; second, epoxidizing the diene compound II under the action of meta-chloroperbenzoic acid to obtain an epoxy compound III; third, brominating the epoxy compound III under the action of hydrobromic acid to obtain a brominated compound IV; and fourth, Suzuki coupling reaction of the brominated compound IV with deuterated methylboronic acid under the catalysis of palladium to obtain deuterated megestrol acetate V. The synthesis method of the present invention is low in cost and mild in conditions. The chemical purity and stable isotope abundance of the obtained deuterated megestrol acetate can reach above 98%, meeting the requirements of a standard reagent for quantitative detection of megestrol acetate compounds and being suitable for industrial production.

The application discloses application of P450 monooxygenase CYP68N3 in catalyzing C11 alpha-hydroxylation of a steroid substrate, wherein the steroid substrate includes 18-methyl nandrolone, 18-methyl diketone, 17-hydroxyprogesterone, canrenone, progesterone and nandrolone, and the like; the expression reaction system of the P450 monooxygenase is optimized, and the whole-cell catalytic efficiency and selectivity are further improved, and the application has important significance for production of C11 alpha-hydroxylation products of various steroid compounds.