CN102864194B - A kind of preparation method of high-purity glucuronic acid bonder - Google Patents
A kind of preparation method of high-purity glucuronic acid bonder Download PDFInfo
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- CN102864194B CN102864194B CN201110188256.6A CN201110188256A CN102864194B CN 102864194 B CN102864194 B CN 102864194B CN 201110188256 A CN201110188256 A CN 201110188256A CN 102864194 B CN102864194 B CN 102864194B
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- BHOPISFXFCYOGY-XFFZJAGNSA-N CC(/C(/C([O]1C=C)=C)=C/C=C)=CC1=O Chemical compound CC(/C(/C([O]1C=C)=C)=C/C=C)=CC1=O BHOPISFXFCYOGY-XFFZJAGNSA-N 0.000 description 1
- YUYBQCUYQYOVKR-WTIBDHCWSA-N COC[C@H](CCC1C=O)OC1C(O)=O Chemical compound COC[C@H](CCC1C=O)OC1C(O)=O YUYBQCUYQYOVKR-WTIBDHCWSA-N 0.000 description 1
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Abstract
A preparation method for high-purity glucuronic acid bonder, is characterized in that: the requirement of described preparation method successively: first carry out bio-conversion processes: be glucuronide conjugate by the conversion of enzyme by substrate conversion; Next carries out separation and purification treatment: the reaction solution obtained by above-mentioned biotransformation is by the enrichment of flash chromatography post and obtain target glucose uronic acid binding substances after purifying.The present invention compares tool with the preparation method of existing glucuronide conjugate and has the following advantages: 1) reaction conditions is gentle, does not need strong acid and strong base; 2) easy and simple to handle, from enrichment in animal body, need not want more purification yet; 3) there is suitability widely, be suitable for the preparation of the glucuronide conjugate of all uridine acyl diphosphate glucose glucuronyl transferase substrates containing phenolic hydroxyl group but not containing acidic-group.
Description
Technical field
The present invention relates to medicine glucuronide conjugate technical field, specifically provide a kind of preparation method of high-purity glucuronic acid bonder.
Background technology
In prior art; glucuronic acid in conjunction with metabolism be a class important drug metabolism reaction; medicine clinically more than 1/3 or its I phase meta-bolites are all the substrates of uridine acyl diphosphate glucose glucuronyl transferase (UGT); O-can be generated in people and animal body; N-; S-, or glucuronide conjugate (the PHarmacol. Ther. 2005 that C-connects; 106 (1): 97-132.).Binding substances is compared with substrate, has higher solubleness, is easy to excrete, thus affects the drug effect of medicine.In addition, some glucuronide conjugate has stronger pharmacologically active or toxicity, and as morphine-6-glucuronide conjugate has stronger anaesthetic effect, estradiol-17-glucuronide conjugate has the ability (Lancet. 1988 of induction cholestasis; 1 (8589): 828, Drug Metab Rev. 1997; 29 (1-2): 183-203.).Therefore, the glucuronide conjugate preparing medicine has very important significance to safe and effective medication clinically.
The method preparing glucuronide conjugate is mainly divided into two classes: chemical method and biological process.Chemical method can realize fairly large preparation, but is not but general class methods, usually needs to develop concrete method for different substrates.In addition, chemical method prepare the reaction conditions that glucuronic acid meta-bolites usually needs the harshness of strong acid and strong base etc., some drug molecule is unstable in these conditions, and the glucuronide conjugate of some drug molecule cannot be prepared by chemical method.Traditionally, the preparation of the glucuronide conjugate of drug molecule mainly obtains from the urine of animal, bile, but this method wastes time and energy, and is difficult to the highly purified glucuronide conjugate of a large amount of acquisition.And binding substances usually with biological sample together, needs multistep separation and purification, could obtain a certain amount of glucuronide conjugate, be difficult to further evaluate drug effect and toxic effect.
Therefore, people expect a kind of method obtaining Efficient preparation of glucose aldehydic acid meta-bolites.
Summary of the invention
The object of this invention is to provide a kind of method of Efficient preparation of glucose aldehydic acid meta-bolites.
The preparation method of a kind of high-purity glucuronic acid bonder of the present invention, the requirement of described preparation method successively:
First bio-conversion processes is carried out: be glucuronide conjugate by the conversion of enzyme by substrate conversion; Enzyme used is specially: recombinant expressed uridine acyl diphosphate glucose glucuronyl transferase (that is: UGT), or for the microsome containing UGT enzymic activity that derives from animal tissues or containing MC enchylema (S9), animal tissues is preferably from the tissue of the strong metabolic capacity such as liver, kidney, intestines; Described substrate is containing phenolic hydroxyl group but not containing the compound of acidic-group;
Next carries out separation and purification treatment: the reaction solution obtained by above-mentioned biotransformation is by the enrichment of flash chromatography post and obtain target glucose uronic acid binding substances after purifying;
Described flash chromatography post filler used is the mixed fillers containing reverse phase silica gel and anionite-exchange resin, and the mass ratio of two kinds of fillers controls at 1-10; Used silica gel is bonded with phenyl, a kind of carbochain in C8 and C18, and silica gel particle diameter, between 10-100 μm, requires containing a kind of basic group in primary amine groups, secondary amine, tertiary amine groups, season amino in anionic ion-exchange resins used.
The preparation method of high-purity glucuronic acid bonder of the present invention, preferred claimed following content:
In bio-conversion processes process, enzyme used requires to use Voranol EP 2001 class nonionic surface active agent (as hexadecanol Soxylat A 25-7) activation in advance, specific requirement is that microsomal protein and promoting agent mass ratio control between 0.1-100, to play maximum catalytic activity.
Biotransformation needs uridine acyl diphosphate glucose aldehydic acid (UDPGA), or is the one in the sodium salt of its correspondence, ammonium salt or sylvite, mole dosage be the 0.1-100 of UGT substrate mole dosage doubly.Above-mentioned substance is intended to for biological catalytic process provides glucal acid groups.
It is carry out in 6-8 buffer salt solution that bioconversion reaction requires at pH value, and keeps the invert point of 30-50 DEG C, preferably uses 35-40 DEG C.
In the filler of described flash chromatography post, silica gel bonded have different lengths carbochain, preferred C8 and C18 of carbon chain lengths, the preferred 40-60 μm of granularity.Anionite-exchange resin is weakly alkaline or strong basic ion exchange resin, and preferably contain the ion exchange resin of primary amine groups or secondary amine group, resin particle diameter is 0.2-1 mm.The total mass of two kinds of fillers is 20 times that are not less than substrate and meta-bolites total mass.
After described bio-transformation sample is placed in flash chromatography post column cap, successively through the process of water wash, alcohol drip washing and the drip washing of acidifying alcohol, water wash is intended to water-soluble substances in elution of reactive liquid, alcohol drip washing is intended to other lipid-soluble substance in wash-out substrate and reaction solution, and the drip washing of acidifying alcohol is intended to wash-out glucuronide conjugate.
Alcohol used is fatty alcohol, concrete nail alcohol, ethanol, Virahol; Acid used is higher boil organic acid, and concrete nail acid, acetic acid, in alcohol, the volumetric molar concentration of acid is 0.01-0.1 mol/L.
In water washing process, the consumption of water is not less than 5 times of column volumes; Alcohol is specially methyl alcohol or ethanol, and the node of alcohol drip washing is no longer containing substrate in elutriant; The consumption of acidifying alcohol is 3-5 times of column volume.
Described bio-transformation sample upper prop and successively by washing, alcohol wash, after acidifying alcohol wash, through high performance liquid chromatography and liquid phase-mass spectrometric detection, meta-bolites is enriched in acidified methanol; Then through low pressure evaporation or rare gas element (N
2) dry up, obtain the meta-bolites of purity >95%.
Glucuronic acid is the reaction mediated by uridine acyl diphosphate glucose glucuronyl transferase (UGT) in conjunction with metabolism, based on chemical method and the traditional biological legal system defect for glucuronide conjugate, the present invention utilizes highly active enzyme, comprise Tooth-Lid Factor T and be derived from animal and there is the microsome of strong metabolic capacity tissue (liver, kidney, intestines etc.) or contain MC enchylema (S9) composition, efficiently prepare grape uronic acid binding substances in vitro.Reaction solution is by containing the flash chromatography post of ion exchange resin and reverse phase filler, the highly purified glucuronide conjugate of disposable acquisition simultaneously.Not only simple to operate, and a large amount of glucuronide conjugates can be obtained.
The present invention compares tool with the preparation method of existing glucuronide conjugate and has the following advantages:
1) reaction conditions is gentle, does not need strong acid and strong base;
2) easy and simple to handle, from enrichment in animal body, need not want more purification yet;
3) there is suitability widely, be suitable for the preparation of the glucuronide conjugate of all UGT substrates containing phenolic hydroxyl group but not containing acidic-group.
accompanying drawing illustrates:
Fig. 1 is high performance liquid chromatography and the mass spectrum of stilboestrol glucuronide conjugate;
Fig. 2 is high performance liquid chromatography and the mass spectrum of magnolol glucuronide conjugate.
embodiment:
The preparation of embodiment 1 stilboestrol glucuronide conjugate, as follows:
Stilboestrol is dissolved in the stock solution being configured to 5 mg/ml in dimethyl sulfoxide (DMSO) (DMSO), taking out 50 ml stock solutions adds in Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) buffered soln of PH7.4, the pork liver microsome (PLM) that nonionogenic tenside (Brij 58) is preactivated is added after stirring, add the donor UDPGA(sodium salt of glucuronic acid), in system, stilboestrol final concentration is 0.25 mg/ml, final concentration of protein is 0.5 mg/ml, UDPGA final concentration 2 mM, 37 DEG C of waters bath with thermostatic control are stirred and are hatched 2-4 h.Reaction solution is transferred to-80 DEG C of refrigerator and cooled but after reaching 90% by high performance liquid chromatography detection substrate transformation efficiency.Extract reaction solution 200 ml and directly go up flash chromatography post.The filler of this flash chromatography post is mixed fillers, and wherein reverse phase silica gel is bonded with C18, and particle diameter is 50 μm, and consumption is 4 g, and ion exchange resin contains primary amino, and materials are 2 g.Successively with 30 ml water wash, 30 ml methyl alcohol drip washing, the carboxylic acid methyl alcohol drip washing of 30 ml.Acidified methanol leacheate rotary evaporation in vacuo at 40 DEG C obtains meta-bolites 77 mg, and HPLC assay products purity >95%(is see Fig. 1).
Embodiment 2, the preparation of magnolol glucuronide conjugate, as follows:
Magnolol is dissolved in the stock solution being made into 5 mg/ml in the middle of methyl alcohol, take out the phosphatic buffering salt that 20 ml add PH7.4, preactivated Tooth-Lid Factor T2B7 is added successively after stirring, add the donor UDPGA(ammonium salt of glucuronic acid), in system, magnolol final concentration is 0.10 mg/ml, final concentration of protein is 0.5 mg/ml, UDPGA final concentration 1 mM, and 37 DEG C of waters bath with thermostatic control are stirred and hatched 1 h.Reaction solution is transferred to-80 DEG C of refrigerator and cooled but after reaching 50% by HPLC detection substrate transformation efficiency.Extract reaction solution 100 ml and directly go up flash chromatography post.The filler of this flash chromatography post is mixed fillers, and wherein silica gel bonded have phenyl, and particle diameter is 40 μm, and consumption is 2.5 g, and ion exchange resin contains secondary amino group, and materials are 2.5 g.Successively with 30 ml water wash, 30 ml methyl alcohol alcohol drip washing, the carboxylic acid methyl alcohol drip washing of 30 ml.Acidified methanol leacheate N under room temperature
2dry up to obtain glucuronide conjugate 9 mg, HPLC purity >95%(Fig. 2).
Embodiment 3, the preparation of Esculetin glucuronide conjugate, as follows:
Esculetin is dissolved in the stock solution being made into 10 mg/ml in the middle of methyl alcohol, take out the buffering salt that 20 ml add the Tris-HCl of PH7.4, preactivated hybrid fine particles body (equivalent people liver particle, pork liver microsome, dog liver particle is added successively after stirring, with rat hepatic microsome mixing), add the donor UDPGA(sylvite of glucuronic acid), in system, Esculetin final concentration is 0.20 mg/ml, and final concentration of protein is 0.4 mg/ml, UDPGA final concentration 1 mM, 37 DEG C of waters bath with thermostatic control are stirred and are hatched 2 h.After HPLC detection substrate transformation efficiency reaches 80%, extract reaction solution 100 ml and directly go up flash chromatography post.The filler of this flash chromatography post is mixed fillers, and wherein reverse phase silica gel is bonded with C8, and particle diameter is 50 μm, and consumption is 2 g, and ion exchange resin contains secondary amino group, and materials are 6 g.Successively with 50 ml water wash, 50 ml methyl alcohol drip washing, 50 ml acetoxylation methyl alcohol drip washing, obtain glucuronide conjugate 16 mg, HPLC purity >95%, glucuronide conjugate nuclear magnetic data is as shown in table 1.
Table 1 Esculetin and the C of its glucuronide conjugate and the chemical shift of H atom
Claims (2)
1. a preparation method for high-purity glucuronic acid bonder, is characterized in that: the requirement of described preparation method successively: first carry out bio-conversion processes: by the conversion of enzyme substrate conversion be glucuronide conjugate and obtain reaction solution to carry out next step operation; Enzyme used is specifically following one of several: the microsome of recombinant expressed uridine acyl diphosphate glucose glucuronyl transferase and UGT, animal tissues containing UGT enzymic activity or containing MC enchylema; Described substrate is containing phenolic hydroxyl group but having certain pharmacologically active and the compound of glucuronic acid in conjunction with metabolism can occur not containing acidic-group;
Next carries out separation and purification treatment: the reaction solution obtained by above-mentioned biotransformation is by the enrichment of flash chromatography post and obtain target glucose uronic acid binding substances after purifying;
Described flash chromatography post filler used is the mixed fillers containing reverse phase silica gel and anionite-exchange resin, and the quality of above-mentioned two kinds of fillers the first and the second is 0.1-10 than ratio; Used silica gel be bonded with several as follows in a kind of group: phenyl, C8, C18, silica gel particle diameter is 10-100 μm; In anionite-exchange resin used containing, for example lower several in a kind of basic group: primary amine groups, secondary amine, tertiary amine groups, season amino group;
In bio-conversion processes process, enzyme used requires to use the activation of Voranol EP 2001 class nonionic surface active agent in advance, and specific requirement is: microsomal protein and promoting agent quality than Ratio control between 0.1-10;
Biotransformation requires to add uridine acyl diphosphate glucose aldehydic acid, or the one added in the sodium salt corresponding with uridine acyl diphosphate glucose aldehydic acid, ammonium salt, sylvite, the mole dosage of uridine acyl diphosphate glucose aldehydic acid or its salt is 0.1-100 times of UGT substrate;
Bioconversion reaction requires to carry out in the buffer salt solution that pH value is 6-8, and keeps the invert point of 30-50 DEG C;
The filler gross weight of described flash chromatography post is 20-100 times of substrate quality;
After bio-transformation sample being placed in flash chromatography post column cap, require successively through water wash, alcohol drip washing and
The process of acidifying alcohol drip washing; Alcohol used is fatty alcohol, and acid used is higher boil organic acid;
Water wash process institute water consumption is not less than 5 times of column volumes, and alcohol lessivation alcohol amount used is not less than 5 times of column volumes, and the amount of acidifying alcohol lessivation acidifying alcohol used is not less than 5 times of column volumes.
2. according to the preparation method of high-purity glucuronic acid bonder described in claim 1, it is characterized in that: fatty alcohol used is the one in methyl alcohol, ethanol and Virahol; Higher boil organic acid used is formic acid or acetic acid.
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| CN101613388A (en) * | 2009-07-16 | 2009-12-30 | 中国科学院大连化学物理研究所 | The coumarin kind compound of glucuronidation and application thereof |
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| CN101613388A (en) * | 2009-07-16 | 2009-12-30 | 中国科学院大连化学物理研究所 | The coumarin kind compound of glucuronidation and application thereof |
Non-Patent Citations (2)
| Title |
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| Identification and Characterization of Human UDP-Glucuronosyltransferases Responsible for the In Vitro Glucuronidation of Daphnetin;Si-Cheng Liang等;《DRUG METABOLISM AND DISPOSITION》;20101231;第38卷(第6期);第973-980页 * |
| 尿苷二磷酸葡萄糖醛酸转移酶的研究进展;彭金咏等;《中国现代应用医学杂志》;20021031;第19卷(第5期);第373-376页 * |
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Effective date of registration: 20180227 Address after: 215600 A 207 room A building center of Zhangjiagang Free Trade Zone, Suzhou Free Trade Zone, Jiangsu Patentee after: Zhangjiagang Institute of industrial technology, Dalian Institute of Chemical Physics, China Academy of Sciences Address before: 116023 Zhongshan Road, Liaoning, No. 457, Patentee before: Dalian Institute of Chemical Physics, Chinese Academy of Sciences |