阅读说明：本技术 一种磺达肝葵钠中间体的合成方法 (Synthetic method of fondaparinux sodium intermediate ) 是由 林蕾 张幸 张立慧 宋萍 黄和 于 2019-08-21 设计创作，主要内容包括：本申请公开一种磺达肝葵钠中间体的合成方法,该方法为化学酶法,从起始糖基受体葡萄糖醛酸的β-对硝基苯苷出发,利用尿苷二磷酸N-三氟乙酰氨基葡萄糖和尿苷二磷酸葡萄糖醛酸在糖基转移酶催化下交替进行糖基化反应构建糖链,然后用酶法进行糖链修饰,最后用碱性裂解和酸催化的苷交换得到目标三糖中间体式(I)。<Image he="303" wi="700" file="DDA0002173961450000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>此方法兼有化学合成的灵活性和生物酶法的高效性,反应无需保护基操作且区域、立体选择性专一,对于降低磺达肝素的成本以及实现大规模生产具有重要的意义。(The application discloses a synthesis method of a fondaparinux sodium intermediate, which is a chemical enzyme method, and the method starts from beta-p-nitrophenyl glycoside of an initial glycosyl acceptor glucuronic acid, uridine diphosphate N-trifluoroacetyl glucosamine and uridine diphosphate glucuronic acid are alternately subjected to glycosylation reaction under the catalysis of glycosyl transferase to construct a sugar chain, then the sugar chain is modified by the enzyme method, and finally alkaline cracking and acid-catalyzed glycoside exchange are carried out to obtain a target trisaccharide intermediate shown in a formula (I). The method has the flexibility of chemical synthesis and the high efficiency of a biological enzyme method, does not need protective group operation in the reaction, has specific regional and stereoselectivity, and has important significance for reducing the cost of fondaparinux and realizing large-scale production.)

1. A synthetic method of fondaparinux sodium intermediate is characterized in that the reaction conditions are as follows:

the first step, starting from the compound (II), introducing trifluoroacetyl glucosamine glycosyl donor through glycosyl transferase catalysis to obtain a compound (III): the compound (II) is an initial reactant, and the using amount is 1.2 mmol; the trifluoroacetyl glucosamine glycosyl donor is uridine diphosphate 2-position trifluoroacetyl glucosamine glycosyl donor, and the using amount of the trifluoroacetyl glucosamine glycosyl donor is 1.5 mmol; the glycosyltransferase is PmHS2, and the dosage is 20 mu g/mL; said compound (II), the 2-position trifluoroacetamido glucosyl donor of uridine diphosphate and glycosyltransferase PmHS2 in Tris and 15mmol MnCl containing 25mmol, pH 7.2₂The reaction was monitored by HPLC for 15 hours at room temperature in the buffer solution of (a), and the product was separated by C18 reverse phase column with a yield of 90% to give compound (III);

secondly, the compound (III) is introduced into a glucuronic acid glycosyl donor through the catalysis of glycosyl transferase to obtain a compound (IV): the glucuronic acid glycosyl donor is uridine diphosphate glucuronyl donor, 1.2mmol of compound (III), 1.5mmol of uridine diphosphate glucuronyl donor and 20 μ g/mL glycosyltransferase PmHS2 are mixed in Tris containing 25mmol, pH 7.2 and 15mmol of MnCl₂The reaction was monitored by HPLC for 15 hours at room temperature in the buffer solution of (a), and the product was separated by C18 reverse phase column with a yield of 91% to give compound (IV);

the third step: repeating the first and second steps, subjecting compound (IV) to glycosyltransferase PmHS2 to catalyze trifluoroacetyl glucosamine glycosyl donor at 91% yield, and then to glycosyltransferase PmHS2 to catalyze glucuronic acid glycosyl donor at 89% yield to obtain compound (V);

in the fourth step, the compound (V) is subjected to alkaline hydrolysis to remove trifluoroacetyl group, and the sulfamide is aminated with a sulfamidase NST to give a compound (VI): adding 1.0equiv of the compound (V) into a 0.1M lithium hydroxide solution, reacting for 2 hours in an ice-water bath, detecting that trifluoroacetyl groups are completely removed by HPLC, adjusting the pH of a reaction system to 7.0 by using 10% dilute hydrochloric acid, adding 50mL of MES solution with pH of 7.0 and 50mM, reacting for 12-15 hours at 37 ℃, and separating a reaction product by using high performance liquid chromatography Q-Sepharose to obtain 0.95g of the compound (VI) with the yield of 96%, wherein 50mL of the MES solution with pH of 7.0 and 50mM contains 10ug/mL of N-sulfotransferase NST and 1mM of PAPS;

the fifth step of subjecting the glucuronic acid structure located at the middle position of the sugar chain of the compound (VI) to racemization at the 5-position of the sugar ring by C5 racemase, and then sulfonating the 2-position of the uronic acid by 2-oxo-sulfatase to obtain a compound (VII): 0.95g, 0.87mmol of Compound (VI) was dissolved in 100mL of MES phosphate buffer solution containing 0.2mM of calcium chloride, 10. mu.g/mL of C5 racemase, 10. mu.g/mL of 2-O-sulfotransferase and 50mM, pH7.0, and reacted at 37 ℃ for 2 hours, and the reaction product was separated by high performance liquid chromatography Q-Sepharose to give 0.82g, 85% yield of Compound (VII);

sixthly, sulfating the 6-position of the sugar ring of the compound (VII) with 6-O-phosphotransferase-1 (6-OST-1) and 6-O-phosphotransferase-3 (6-OST-3) to obtain a compound (VIII): 0.82g, 0.74mmol of compound (VII) was dissolved in 100mL of 50M MES phosphate buffer solution containing 1.5mM 3 '-phosphoadenosine-5' -phosphosulfate, 0.2. mu.g/mL sulfotransferase 6-OST-1, 0.2. mu.g/mL sulfotransferase 6-OST-3, pH7, and reacted at 37 ℃ for 15 hours, and the reaction product was isolated by high performance liquid chromatography Q-Sepharose to give 0.75g, 92% yield of compound (VIII) as a white solid;

the seventh step: degrading the compound (VIII) by using alkalinity, and cracking glucuronic acid parts at two ends of a sugar chain to obtain a compound (IX);

eighth step: dissolving the compound (IX) in 0.05mM absolute methanol hydrochloride solution at room temperature, and performing glycoside exchange by acid catalysis to obtain alpha-methyl glycoside, namely the formula (I).

2. A synthetic method of fondaparinux sodium intermediate is characterized by comprising the following steps: and in the first step, the second step and the third step, the trifluoroacetylaminoglucose radical donor and the glucuronic acid radical donor are in a diphosphonic acid form or a diphosphonic acid form, and specifically are sodium salt, potassium salt, ammonium salt, triethylamine salt or pyridine salt.

3. The method for synthesizing fondaparinux sodium intermediate according to claim 2, wherein: the method of alkaline degradation in the seventh step is to dissolve 1g, 0.9mmol of the compound (VIII) in 10mL of NaH with pH7.0₂PO₄Adding 20equiv of sodium periodate into a buffer solution, reacting for 3.5 hours at 37 ℃, then adding 20equiv of ethylene glycol to quench the reaction, intercepting the molecular weight of the reaction solution by a dialysis bag and desalting, freeze-drying, adding 10mL of 0.5M sodium hydroxide solution, reacting for 3 hours at normal temperature, intercepting the molecular weight of the product by the dialysis bag and desalting, and freeze-drying to obtain 0.7g of a compound (IX) with the yield of 90%.

4. The method for synthesizing fondaparinux sodium intermediate according to claim 3, wherein: the eighth step was carried out using acid catalysis for glycoside exchange to produce α -methylglycoside, and specifically comprises adding lyophilized compound (IX) to 100mL of 0.05mM methanol hydrochloride anhydrous solution, stirring for 1 hour, adding 0.05mM NaOH solution to neutralize to pH7.0, and separating by P-2 gel column to obtain 0.65g of formula (I) with 90% yield.

5. The method for synthesizing fondaparinux sodium intermediate according to claim 3, wherein: the glycosyltransferase in the first, second and third steps is PmHS2, the sulfamidase in the fourth step is NST, the C5 racemase in the fifth step is C5-epi, the 2-O-phosphotransferase in the fifth step is 2-OST, and the sulfatase in the sixth step is 6-O-phosphotransferase-1 (6-OST-1) and 6-O-phosphotransferase-3 (6-OST-3).

6. The method for synthesizing fondaparinux sodium intermediate according to claim 3, wherein: the fourth sulfonamide step can be achieved either using the sulfonamide transferase NST or by a chemical method which is: 1.0equiv of Compound (V) and 3.0equiv of Sulfur trioxide-trimethylamine complex were dissolved in 5vol of DMF and reacted at 37 ℃ for 5 hours.

Technical Field

The application relates to the technical field of medicines, in particular to a synthesis method of a fondaparinux sodium intermediate.

Background

Fondaparinux sodium (Fondaparinux sodium) under the trade name Fondaparinux sodium

Is the only heparin oligosaccharide anticoagulant drug which is chemically synthesized at present and is developed and produced by SanofiWinthrop Industrial in France,CAS number 114870-03-0. This is an ultra-low molecular heparin synthesized based on the structure of the action site core pentasaccharide in which heparin specifically binds to Antithrombin III (AT III), and the structure is as follows:

compared with unfractionated and low molecular weight heparin, fondaparinux can greatly reduce the incidence of heparin-induced thrombocytopenia, and is therefore safer to use clinically. However, the chemical synthesis of flava heparin involves various protecting group manipulations and selective sulfation of different types, the reaction steps are multiple, the separation and purification are difficult, the synthesis efficiency is low, for example, the total synthesis steps are as many as 60 steps, and the total yield is less than 0.1%, which also causes that flava heparin is the most expensive heparin drug, and the wider application of flava heparin is greatly limited. The method has the advantages of chemical enzyme synthesis, simulation of a biological synthesis way of heparin, flexibility of chemical synthesis and high efficiency of a biological enzyme method, no need of protecting group operation in reaction, and specificity in regional and stereoselectivity, and has important significance for reducing the cost of fondaparinux and realizing large-scale production.

Particularly, the trisaccharide intermediate of fondaparinux, shown in formula (I), contains polysulfate group, iduronic acid and alpha-methyl glycoside at the reducing end, and is the main difficulty and core part of the existing fondaparinux production. Therefore, development of a synthetic method for this intermediate is required.

Content of application

The technical problem to be solved is as follows:

the technical problems to be solved by the application are that the reaction steps are large, the separation and purification are difficult, the synthesis efficiency is low and the like in the prior art, and the synthesis method of the fondaparinux sodium intermediate is provided.

The technical scheme is as follows:

a synthetic method of fondaparinux sodium intermediate comprises the following reaction conditions:

the first step, starting from the compound (II), introducing trifluoroacetyl glucosamine glycosyl donor through glycosyl transferase catalysis to obtain a compound (III): the compound (II) is an initial reactant, and the using amount is 1.2 mmol; the trifluoroacetyl glucosamine glycosyl donor is uridine diphosphate 2-position trifluoroacetyl glucosamine glycosyl donor, and the using amount of the trifluoroacetyl glucosamine glycosyl donor is 1.5 mmol; the glycosyltransferase is PmHS2, and the dosage is 20 mu g/mL; said compound (II), the 2-position trifluoroacetamido glucosyl donor of uridine diphosphate and glycosyltransferase PmHS2 in Tris and 15mmol MnCl containing 25mmol, pH 7.2₂The reaction was monitored by HPLC for 15 hours at room temperature in the buffer solution of (a), and the product was separated by C18 reverse phase column with a yield of 90% to give compound (III);

secondly, the compound (III) is introduced into a glucuronic acid glycosyl donor through the catalysis of glycosyl transferase to obtain a compound (IV): the glucuronic acid glycosyl donor is uridine diphosphate glucuronyl donor, 1.2mmol of compound (III), 1.5mmol of uridine diphosphate glucuronyl donor and 20 μ g/mL glycosyltransferase PmHS2 are mixed in Tris containing 25mmol, pH 7.2 and 15mmol of MnCl₂The reaction was monitored by HPLC for 15 hours at room temperature in the buffer solution of (a), and the product was separated by C18 reverse phase column with a yield of 91% to give compound (IV);

the third step: repeating the first and second steps, subjecting compound (IV) to glycosyltransferase PmHS2 to catalyze trifluoroacetyl glucosamine glycosyl donor at 91% yield, and then to glycosyltransferase PmHS2 to catalyze glucuronic acid glycosyl donor at 89% yield to obtain compound (V);

in the fourth step, the compound (V) is subjected to alkaline hydrolysis to remove trifluoroacetyl group, and the sulfamide is aminated with a sulfamidase NST to give a compound (VI): adding 1.0equiv of the compound (V) into a 0.1M lithium hydroxide solution, reacting for 2 hours in an ice-water bath, detecting that trifluoroacetyl groups are completely removed by HPLC, adjusting the pH of a reaction system to 7.0 by using 10% dilute hydrochloric acid, adding 50mL of MES solution with pH7.0 and 50mM, reacting for 12-15 hours at 37 ℃, and separating a reaction product by using high performance liquid chromatography Q-Sepharose to obtain 0.95g of the compound (VI) with the yield of 96%, wherein 50mL of the MES solution with pH7.0 and 50mM contains 10ug/mL of N-sulfotransferase NST and 1mM of PAPS;

the fifth step of subjecting the glucuronic acid structure located at the middle position of the sugar chain of the compound (VI) to racemization at the 5-position of the sugar ring by C5 racemase, and then sulfonating the 2-position of the uronic acid by 2-oxo-sulfatase to obtain a compound (VII): 0.95g, 0.87mmol of Compound (VI) was dissolved in 100mL of MES phosphate buffer solution containing 0.2mM of calcium chloride, 10. mu.g/mL of C5 racemase, 10. mu.g/mL of 2-O-phosphotransferase and 50mM, pH7.0, and reacted at 37 ℃ for 2 hours. The reaction product was separated by high performance liquid chromatography Q-Sepharose to give 0.82g of compound (VII) in 85% yield;

sixthly, sulfating the 6-position of the sugar ring of the compound (VII) with 6-O-phosphotransferase-1 (6-OST-1) and 6-O-phosphotransferase-3 (6-OST-3) to obtain a compound (VIII): 0.82g, 0.74mmol of compound (VII) was dissolved in 100mL50M MES phosphate buffer solution containing 1.5mM 3 '-phosphoadenosine-5' -phosphosulfate, 0.2. mu.g/mL sulfotransferase 6-OST-1, 0.2. mu.g/mL sulfotransferase 6-OST-3, pH7, and reacted at 37 ℃ for 15 hours, and the reaction product was isolated by high performance liquid chromatography Q-Sepharose to give compound (VIII) as a white solid in 0.75g, 92% yield;

the seventh step: degrading the compound (VIII) by using alkalinity, and cracking glucuronic acid parts at two ends of a sugar chain to obtain a compound (IX); eighth step: dissolving the compound (IX) in 0.05mM absolute methanol hydrochloride solution at room temperature, and performing glycoside exchange by acid catalysis to obtain alpha-methyl glycoside, namely the formula (I).

As a preferred technical scheme of the application: and in the first step, the second step and the third step, the trifluoroacetylaminoglucose radical donor and the glucuronic acid radical donor are in a diphosphonic acid form or a diphosphonic acid form, and specifically are sodium salt, potassium salt, ammonium salt, triethylamine salt or pyridine salt.

As a preferred technical scheme of the application: the method of alkaline degradation in the seventh step is to dissolve 1g, 0.9mmol of the compound (VIII) in 10mL of NaH with pH7.0₂PO₄Adding 20equiv of sodium periodate into a buffer solution, reacting for 3.5 hours at 37 ℃, then adding 20equiv of ethylene glycol to quench the reaction, intercepting the molecular weight of the reaction solution by a dialysis bag and desalting, freeze-drying, adding 10mL of 0.5M sodium hydroxide solution, reacting for 3 hours at normal temperature, intercepting the molecular weight of the product by the dialysis bag and desalting, and freeze-drying to obtain 0.7g of a compound (IX) with the yield of 90%.

As a preferred technical scheme of the application: the eighth step was carried out using acid catalysis for glycoside exchange to produce α -methylglycoside, and specifically comprises adding lyophilized compound (IX) to 100mL of 0.05mM methanol hydrochloride anhydrous solution, stirring for 1 hour, adding 0.05mM NaOH solution to neutralize to pH7.0, and separating by P-2 gel column to obtain 0.65g of formula (I) with 90% yield.

As a preferred technical scheme of the application: the glycosyltransferase in the first, second and third steps is PmHS2, the sulfamidase in the fourth step is NST, the C5 racemase in the fifth step is C5-epi, the 2-O-phosphotransferase in the fifth step is 2-OST, and the sulfatase in the sixth step is 6-O-phosphotransferase-1 (6-OST-1) and 6-O-phosphotransferase-3 (6-OST-3).

As a preferred technical scheme of the application: the fourth sulfonamide step can be achieved either using the sulfonamide transferase NST or by a chemical method which is: 1.0equiv of Compound (V) and 3.0equiv of Sulfur trioxide-trimethylamine complex were dissolved in 5vol of DMF and reacted at 37 ℃ for 5 hours.

Has the advantages that:

compared with the prior art, the synthesis method of the fondaparinux sodium intermediate has the following technical effects:

1. the method has the advantages of combining the flexibility of chemical synthesis and the high efficiency of a biological enzyme method, having no need of protecting group operation in the reaction and specific regioselectivity and stereoselectivity, and having important significance for reducing the cost of fondaparinux and realizing large-scale production;

2. the synthesis of fondaparinux by the prior art needs to use a large amount of organic metal reagents for glycosylation coupling, so that the environmental pollution is caused, and the invention avoids the use of toxic reagents;

3. the existing process involves a large amount of low-temperature reaction (-78 ℃) and anhydrous anaerobic reaction protected by inert gas for the synthesis of fondaparinux, which is not beneficial to large-scale operation, and the reaction condition of the synthetic route of the invention is basically room temperature to 37 ℃, and the anhydrous anaerobic reaction does not exist;

4. the synthesis of the compound (I) by the route of the prior art needs more than 30 steps, and the invention only needs 8 steps.

Detailed Description

The technical scheme of the invention is further explained in detail as follows:

it will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the prior art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

Compound (II) was purchased from Wuhanxin Jiali Biotech, Inc., model 20140201.