CN108866141A - A kind of enzymatic synthesis method of ursodesoxycholic acid - Google Patents
A kind of enzymatic synthesis method of ursodesoxycholic acid Download PDFInfo
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Abstract
Disclosed by the invention is a kind of enzymatic synthesis method of ursodesoxycholic acid, when solving in the prior art using chenodeoxycholic acid preparation ursodesoxycholic acid, usually occurs the case where 3 hydroxyls of simultaneous oxidation in oxidation-reduction process, the problem for causing yield and purity not high.The present invention includes step 1, and the 7-KLCA as shown in formula one is made；Step 2,7-KLCA is added in phosphate buffer, reaction solution is made, L MALIC ACID sodium, malic dehydrogenase, 7- β steroid dehydrogenase are added in reaction solution, control reaction temperature 25~30 DEG C, reaction pH be 7.0~7.5 under the conditions of, reaction until 7-KLCA residual be lower than 0.1%, after the reaction was completed, add alkali to be warming up to 60~80 DEG C to stir 1~3 hour, then it is cooled to room temperature, is filtered, filtrate acid adding crystallizes to obtain ursodesoxycholic acid.The advantages such as the present invention has yield and purity is high, and production cost is low.
Description
Technical field
The present invention relates to a kind of synthetic methods, and in particular to be a kind of ursodesoxycholic acid enzymatic synthesis method.
Background technique
Chenodeoxycholic acid is the raw material for preparing ursodesoxycholic acid, in the technique using chenodeoxycholic acid preparation ursodesoxycholic acid
In, ursodesoxycholic acid is to obtain 5 β -3 Alpha-hydroxy -7- carbonyl cholanic acid (7K- with its 7 epimer chenodeoxycholic acid oxidases
LCA), restore to synthesize.Conventionally, as containing in the structure of chenodeoxycholic acid, there are two hydroxyls, in preparation process
In, the case where being theoretically oxidized in the presence of one or two.And industry technical staff be usually use NBS, PCC,
The substances such as chromium trioxide aoxidize chenodeoxycholic acid, and 7- ketone group stone gallbladder is realized using metallic sodium or Pd/C catalytic hydrogenation
The reduction of sour (7-KLCA).
In the prior art, although above-mentioned mode of oxidizing has achieved the purpose that 7 hydroxyls are oxidized, but simultaneously also to portion
3 hydroxyls are divided to be aoxidized, since the case where researcher of this field is individually oxidized for 3 and 7 research is very few,
The independent mode of oxidizing of especially 7 hydroxyls, although there is document report, in acetone and water oxygen CDCA, 5 β-to be made using NBS
3 Alpha-hydroxy -7- carbonyl cholanic acids (7K-LCA), yield is up to 89%.But final purity is not high, aoxidizes in manufactured finished product
The case where there is also part two while being oxidized.
There is also selectivity is low in the method for above-mentioned reduction, industrial amplification production is not easily controlled and unsafe asks
Topic.
Summary of the invention
When it is an object of the invention to solve in the prior art using chenodeoxycholic acid preparation ursodesoxycholic acid, gone back in oxidation
Usually there is the case where 3 hydroxyls of simultaneous oxidation during former, the problem for causing yield and purity not high；It provides and solves above-mentioned ask
A kind of enzymatic synthesis method of ursodesoxycholic acid of topic.
In order to achieve the above objectives, technical scheme is as follows：
A kind of enzymatic synthesis method of ursodesoxycholic acid, including：
Step 1, the 7-KLCA as shown in formula one is made；
Formula one：
Step 2,7-KLCA is added in phosphate buffer and reaction solution is made, in reaction solution be added L MALIC ACID sodium,
Malic dehydrogenase, 7- β steroid dehydrogenase, control reaction temperature 25~30 DEG C, reaction pH be 7.0~7.5 under the conditions of, instead
0.1% should be lower than until 7-KLCA is remained, after the reaction was completed, add alkali to be warming up to 60~80 DEG C and stir 1~3 hour, then cool down
To room temperature, filtering, filtrate acid adding crystallizes to obtain ursodesoxycholic acid；
Wherein, the specific preparation step of step 1 includes：
Step 1.1, chenodeoxycholic acid is dissolved in methanol solvate and mixed solution is made, sodium bromide is added into mixed solution
It is configured to electrolysis system；
Step 1.2, electrolysis system is added in the undivided cell of graphite electrode, the 10~12h that is persistently powered is obtained
Oxidation product, electric current is 3~5A in undivided cell, and reaction temperature is -5~5 DEG C；
Step 1.3, dilution heat of sulfuric acid is added dropwise into oxidation product, reacts 30min or more, reaction temperature is 20~60 DEG C；
The gas that reaction generates, which extend into react in NaOH solution with NaOH solution, is made sodium bromide；
Step 1.4,7-KLCA is made in purification.
By above-mentioned setting, it can effectively realize that 7 hydroxyls individually aoxidize completely, 3 hydroxyls will not be aoxidized, reach selectivity
The purpose of oxidation, improves purity and yield, effect are very significant.
The present invention can not only achieve the purpose that selective oxidation, moreover, the bromo element in the present invention can repeat to make
With, that is, low-boiling hydrogen bromide can effectively be displaced using high boiling dilution heat of sulfuric acid, the hydrogen bromide displaced is added
Into NaOH solution, raw material sodium bromide can also be made again by drying, and then achieve the purpose that recycling, production cost
It is lower.
Further, the molar ratio of the sodium bromide in the step 1.2 and the dilute sulfuric acid of step 3 kind is 2.1~2.3 ︰ 1.Institute
The reaction temperature for stating step 1.2 is 0~5 DEG C.The reaction temperature of the step 1.3 is 40~50 DEG C.
In order to reach better purity, the method for the purification is：Solution after filtering step 1.3 reaction, is washed with pure water
It washs, liquid separation, dry, finished product is made in reduced pressure.
By above-mentioned setting, better yield and purity can be effectively obtained, yield can achieve 95% or more, and purity can
To reach 98% or more, effect is very significant.
Further, the acid in the step 2 is inorganic acid, and crystallization pH is 2~3.L MALIC ACID sodium dosage in the step 2
It is the 50~60% of 7-KLCA weight；Malate dehydrogenase enzyme dosage is the 1.5~2.2% of 7-KLCA weight；7- β steroid dehydrogenase
Enzyme dosage is the 0.1~0.3% of 7-KLCA weight.
Compared with prior art, the present invention having the following advantages that and beneficial effect：
1, the present invention can effectively realize that 7 hydroxyls individually aoxidize completely, will not aoxidize 3 hydroxyls, reach selective oxidation
Purpose；
2, the bromo element in the present invention may be reused, that is, can effectively be replaced using high boiling dilution heat of sulfuric acid
The hydrogen bromide displaced is added in NaOH solution by low-boiling hydrogen bromide out, and raw material can also be made again by drying
Sodium bromide, and then achieve the purpose that recycling, production cost is lower；
3, yield of the invention can achieve 90% or more, and purity can achieve 98% or more, and effect is very significant.
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail, embodiments of the present invention are not limited thereto.
Embodiment 1
A kind of enzymatic synthesis method of ursodesoxycholic acid, including：
Step 1, the 7-KLCA as shown in formula one is made；
Formula one：
Step 2,100g 7-KLCA is added in 500mL phosphate buffer and reaction solution is made, be added in reaction solution
55g L MALIC ACID sodium, 2g malic dehydrogenase, 0.2g 7- β steroid dehydrogenase, control reaction temperature 25~30 DEG C, it is anti-
Answer pH be 7.0~7.5 under the conditions of, reaction until 7-KLCA residual be lower than 0.1%, after the reaction was completed, add mass fraction be 30%
NaOH solution 10g be warming up to 70 DEG C stir 2 hours, be then cooled to room temperature, filter, filtrate add inorganic acid until pH reach 2
~3, ursodesoxycholic acid is obtained after crystallization.
Wherein, the specific preparation step of step 1 includes：
Step 1.1,100g chenodeoxycholic acid is dissolved in 1000mL methanol solvate and mixed solution is made, into mixed solution
Sodium bromide 1.03g is added and is configured to electrolysis system；
Step 1.2, electrolysis system is added in the undivided cell of graphite electrode, persistently energization 12h is aoxidized
Product, electric current is 4A in undivided cell, and reaction temperature is 2 DEG C；
Step 1.3, the dilution heat of sulfuric acid that dropwise addition 7.19g mass fraction is 30% into oxidation product, reaction time 1h,
Reaction temperature is 45 DEG C；The gas that reaction generates is passed into the bottom end of NaOH solution, reacts gas with NaOH solution and bromine is made
Change sodium solution, raw material sodium bromide is made after sodium bromide solution is dry；
Step 1.4,7-KLCA described in formula one is made in purification.
Above-mentioned finished product is detected, testing result is shown：The yield of 7-KLCA has reached 97%, purity and has reached
99%, the yield of ursodesoxycholic acid reaches 90%, purity and has reached 98%, and effect is very significant.
Embodiment 2
The present embodiment the difference from embodiment 1 is that, the response parameter of step 1 is different in the present embodiment, and specific setting is such as
Under：
Step 1.1,200g chenodeoxycholic acid is dissolved in 1500mL methanol solvate and mixed solution is made, into mixed solution
Sodium bromide 2.10g is added and is configured to electrolysis system；
Step 1.2, electrolysis system is added in the undivided cell of graphite electrode, persistently energization 12h is aoxidized
Product, electric current is 3A in undivided cell, and reaction temperature is 5 DEG C；
Step 1.3, the dilution heat of sulfuric acid that dropwise addition 15.42g mass fraction is 30% into oxidation product, reaction time are
1h, reaction temperature are 40 DEG C；
Step 1.4, it filters, with pure water, liquid separation is dry, and 7-KLCA described in formula one is made in reduced pressure.
Above-mentioned finished product is detected, testing result is shown：The yield of 7-KLCA has reached 95%, and purity reaches
98%.
Embodiment 3
The present embodiment the difference from embodiment 1 is that, the response parameter of step is different in the present embodiment, and specific setting is such as
Under：
Step 1.1,100g chenodeoxycholic acid is dissolved in 1000mL methanol solvate and mixed solution is made, into mixed solution
Sodium bromide 1.10g is added and is configured to electrolysis system；
Step 1.2, electrolysis system is added in the undivided cell of graphite electrode, persistently energization 12h is aoxidized
Product, electric current is 5A in undivided cell, and reaction temperature is 0 DEG C；
Step 1.3, the dilution heat of sulfuric acid that dropwise addition 9.42g mass fraction is 30% into oxidation product, reaction time 1h,
Reaction temperature is 35 DEG C；
Step 1.4, it filters, with pure water, liquid separation is dry, and 7-KLCA described in formula one is made in reduced pressure.
Above-mentioned finished product is detected, testing result is shown：The yield of 7-KLCA has reached 91%, and purity reaches
95%.
Above-described embodiment is merely a preferred embodiment of the present invention, and it is not intended to limit the protection scope of the present invention, as long as using
Design principle of the invention, and the non-creative variation worked and made is carried out on this basis, it should belong to of the invention
Within protection scope.
Claims (7)
1. a kind of enzymatic synthesis method of ursodesoxycholic acid, which is characterized in that including：
Step 1, the 7-KLCA as shown in formula one is made；
Formula one：
Step 2,7-KLCA is added in phosphate buffer and reaction solution is made, L MALIC ACID sodium, apple are added in reaction solution
Acidohydrogenase, 7- β steroid dehydrogenase, control reaction temperature 25~30 DEG C, reaction pH be 7.0~7.5 under the conditions of, reaction is straight
It is lower than 0.1% to 7-KLCA residual, after the reaction was completed, adds alkali to be warming up to 60~80 DEG C and stir 1~3 hour, be then cooled to often
Temperature, filtering, filtrate acid adding crystallize to obtain ursodesoxycholic acid；
Wherein, the specific preparation step of step 1 includes：
Step 1.1, chenodeoxycholic acid is dissolved in methanol solvate and mixed solution is made, sodium bromide configuration is added into mixed solution
At electrolysis system；
Step 1.2, electrolysis system is added in the undivided cell of graphite electrode, the 10~12h that is persistently powered is aoxidized
Product, electric current is 3~5A in undivided cell, and reaction temperature is -5~5 DEG C；
Step 1.3, dilution heat of sulfuric acid is added dropwise into oxidation product, reacts 30min or more, reaction temperature is 20~60 DEG C；Reaction
The gas of generation, which extend into react in NaOH solution with NaOH solution, is made sodium bromide；
Step 1.4,7-KLCA is made in purification.
2. a kind of enzymatic synthesis method of ursodesoxycholic acid according to claim 1, which is characterized in that the step 1.2
In sodium bromide and step 3 kind dilute sulfuric acid molar ratio be 2.1~2.3 ︰ 1.
3. a kind of enzymatic synthesis method of ursodesoxycholic acid according to claim 1, which is characterized in that the step 1.2
Reaction temperature be 0~5 DEG C.
4. according to a kind of described in any item enzymatic synthesis methods of ursodesoxycholic acid of Claims 2 or 3, which is characterized in that institute
The reaction temperature for stating step 1.3 is 40~50 DEG C.
5. a kind of enzymatic synthesis method of ursodesoxycholic acid according to claim 1, which is characterized in that the side of the purification
Method is：Solution after filtering step 1.3 reaction, with pure water, liquid separation is dry, and 7-KLCA is made in reduced pressure.
6. a kind of enzymatic synthesis method of ursodesoxycholic acid according to claim 1, which is characterized in that in the step 2
Acid be inorganic acid, crystallization pH be 2~3.
7. a kind of enzymatic synthesis method of ursodesoxycholic acid according to claim 1, which is characterized in that in the step 2
L MALIC ACID sodium dosage is the 50~60% of 7-KLCA weight；Malate dehydrogenase enzyme dosage be 7-KLCA weight 1.5~
2.2%；7- β steroid dehydrogenase enzyme dosage is the 0.1~0.3% of 7-KLCA weight.
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