CN105001140B - A kind of method and its application for preparing fat diacid derivative - Google Patents
A kind of method and its application for preparing fat diacid derivative Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D207/46—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with hetero atoms directly attached to the ring nitrogen atom
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/62—Insulins
Abstract
The invention discloses a kind of method and its application for preparing fat diacid derivative.This method is first to react long-chain fat diacid and thionyl chloride, obtains acyl chlorides；Acyl chlorides and the tert-butyl alcohol are reacted, then remove cl radical, obtains the tert-butyl ester of long-chain fat diacid one；Then reacted with N HOSu NHSs, long-chain fat diacid succinimido tertiary butyl ester；Then reacted with the tert-butyl ester of L glutamic acid 1, obtain tert-butyl group long-chain fat diacyl L Glu OtBu；Reacted again with N HOSu NHSs, obtain the tert-butyl group long-chain fat diacyl L Glu (OSu) OtBu；Finally the tert-butyl group in the tert-butyl group long-chain fat diacyl L Glu (OSu) OtBu is removed, obtains long-chain fat diacid derivative.The route of this method is easy to operate, quality controllable, is adapted to industry's enlarging production, and cost is low, and Simultaneous purification is simple, is suitable for preparing the insulin analog of high-purity.
Description
Technical field
The present invention relates to biomedicine technical field, more particularly to a kind of method for preparing fat diacid derivative and its should
With.
Background technology
Fat diacid derivative plays in the research of third generation insulin medicament especially Recent Development of Long-acting Insulin Analogs
Important function, insulin glargine reached 72.79 as the weight pound medicine of Recent Development of Long-acting Insulin Analogs in global marketing volume in 2014
Hundred million dollars.However, insulin glargine, which is difficult to reach, smoothly controls blood glucose and stable pharmacokinetic profile, directly result in low
Blood glucose incidence especially Nocturnal hypoglycemia incidence is higher, threat to life.
The research as the Recent Development of Long-acting Insulin Analogs of insulin glargine alternative medicine is increasingly becoming diabetes and controlled in recent years
The focus in treatment field, wherein Publication No. CN1829738, the Chinese invention patent of entitled " novel insulin derivates " disclose
Recent Development of Long-acting Insulin Analogs --- the moral paddy insulin that fat diacid side chain is modified by glutamic acid；Publication No.
CN102065884, the Chinese invention patent of entitled " insulin lispro compounds of Pegylation " disclose molecular weight about
The conjugated insulin analog of 20kDa polyethylene glycol；Publication number WO 2011/122921 PCT Application Publication and immune ball
The insulin conjugate that albumen Fc fragments are coupled by PEG.Thibaudeau and Bridon discloses seralbumin coupling
Insulin conjugate (Thibaudeau and Bridon et al., Bioconjugate chemistry, 2005,16,1000-
1008), author claims that the conjugate can turn into potential Recent Development of Long-acting Insulin Analogs candidate medicine in article.
In many Recent Development of Long-acting Insulin Analogs research, moral paddy insulin half-life period is most long, and internal action time is more than 42
Hour, while risk of hypoglycemia especially Nocturnal hypoglycemia risk is low, most promises to be another after insulin glargine
Weight pound medicine.The molecular structural formula of moral paddy insulin (Degludec, trade name Tresiba) shown in formula I, its molecular structure
Feature is that long-chain fat diacid is connected on 29 lysine residues of insulin B by a glutamate molecule.
The preparation of long-chain fat diacid derivative and modification reaction become important in insulin analog preparation technology
Link.N is employed in the tert-butyl ester preparation technology of hexadecandioic acid (hexadecane diacid) one disclosed in Novo Nordisk Co., Ltd patent No. CN1829738,
For dinethylformamide di-t-butyl acetal as tert-butyl ester reagent, cost is high, is not suitable for industry amplification；Hexadecandioic acid (hexadecane diacid) amber
2- succinimido -1,1,3,3- tetramethylurea tetrafluoro boric acid esters are employed in amber imide tertiary butyl ester preparation technology
(TSTU), cost is high, is not suitable for industrial amplification production；Hexadecandioic acid (hexadecane diacid) mono-methyl derivative is which disclose simultaneously, by
The piptonychia ester in 0.2M NaOH solutions is needed in insulin analog prepared by the technique, easily causes point of insulin peptide chain
Solution, impurity is more, is equally difficult to industrial amplification production.Grinstaff and Zhang disclose DMAP
(DMAP) dicyclohexylcarbodiimide (DCC) condensation dodecanedioic acid and the tert-butyl alcohol are catalyzed, prepares the tert-butyl ester of dodecanedioic acid one
Technique (Grinstaff and Zhang et al., Bioconjugate chemistry, 2011,22 (4), 690-9), and with the work
The accessory substance hexadecandioic acid (hexadecane diacid) N- acylureas ratio that skill prepares the generation of the tert-butyl ester of hexadecandioic acid (hexadecane diacid) one is high, it is difficult to separated with product,
It is also not suitable for technique amplification production.
The content of the invention
The shortcomings that primary and foremost purpose of the present invention is to overcome prior art and deficiency, there is provided one kind prepares fat diacid derivative
The method of thing.This method is easy to operate, product related impurities content is low, purification of intermediate is easy, high income.Prepared fat
Diacid derivative can be used for the research and development of protamine zine insulin medicine such as moral paddy insulin by protein modified general technology
Another object of the present invention is to provide the application of the method for preparing fat diacid derivative.
The purpose of the present invention is achieved through the following technical solutions：A kind of method for preparing fat diacid derivative, including such as
Lower step：
(1) the long-chain fat diacid as shown in formula A and thionyl chloride are reacted, obtains carrying two chlorine as shown in formula B
The acyl chlorides of group；Then acyl chlorides and the tert-butyl alcohol are reacted, obtains the ester with a cl radical as shown in formula C；Then hydroxyl is used
Base substitutes cl radical, obtains the tert-butyl ester of long-chain fat diacid one as shown in formula D；Wherein, the m in following reaction equations is 6
~32 integer, preferably 6~9；
(2) tert-butyl ester of long-chain fat diacid one and n-hydroxysuccinimide are reacted, obtains the long-chain fat as shown in formula E
Fat diacid succinimido tertiary butyl ester；
(3) long-chain fat diacid succinimido tertiary butyl ester and the Pidolidone -1- tert-butyl esters are reacted, obtained such as formula
Tert-butyl group long-chain fat diacyl-L-Glu-OtBu shown in F；
(4) tert-butyl group long-chain fat diacyl-L-Glu-OtBu and n-hydroxysuccinimide are reacted, obtained such as formula G
Shown tert-butyl group long-chain fat diacyl-L-Glu (OSu)-OtBu；
(5) tert-butyl group in tert-butyl group long-chain fat diacyl-L-Glu (OSu)-OtBu is removed, obtained as shown in formula H
Long-chain fat diacid derivative.
The dosage of thionyl chloride described in step (1) is excess, the i.e. mol ratio of thionyl chloride and long-chain fat diacid
More than 2；Preferably 6~23.6：1.
The dosage of the tert-butyl alcohol described in step (1) is 0.6~1.5 by the tert-butyl alcohol and long-chain fat diacid mole ratio：1；
Preferably 1~1.2：1 calculates；More preferably press 1.1：1 calculates.
Described in step (1) is by by described ester and solvent with a cl radical with hydroxyl substitution cl radical
Water reaction in the A aqueous solution；Solvent orange 2 A is for one kind in N,N-dimethylformamide (DMF), acetonitrile, acetone and tetrahydrofuran or extremely
Two kinds, preferably DMF less.
The volume ratio of solvent orange 2 A and water is 1 in the described solvent orange 2 A aqueous solution：1~5, preferably 1：2~3, more preferably 1：
2。
The dosage of n-hydroxysuccinimide described in step (2) presses itself and the described tert-butyl ester of long-chain fat diacid one
Mol ratio is 1~1.2：1 calculates, preferably by 1.05：1 calculates.
The dosage of the Pidolidone -1- tert-butyl esters described in step (3) presses itself and described long-chain fat diacid succinyl
Imido grpup tertiary butyl ester mol ratio is 1~1.5：1 calculates, preferably by 1~1.2：1 calculates, more preferably by 1.1：1 calculates.
The dosage of n-hydroxysuccinimide described in step (4) presses itself and the described acyl of tert-butyl group long-chain fat two
Base-L-Glu-OtBu mol ratios are 1~1.2：1 calculates, preferably by 1.1：1 calculates.
Described in step (5) by tert-butyl group long-chain fat diacyl-L-Glu (OSu)-OtBu the tert-butyl group remove be
By the way that described tert-butyl group long-chain fat diacyl-L-Glu (OSu)-OtBu and trifluoroacetic acid are reacted.
The dosage of described trifluoroacetic acid presses itself and described tert-butyl group long-chain fat diacyl-L-Glu (OSu)-OtBu
Volume mass ratio is 3~10mL: 1g calculating；Preferably calculated by 3~5mL: 1g；More preferably calculated by 5mL: 1g.
In order to reduce the impurity of whole course of reaction, increase the yield of product and improve the purity of product, often obtaining one
During individual product, the material for having neither part nor lot in reaction is removed.
So as to the described method for preparing fat diacid derivative, preferably include following steps：
1) acyl chlorides is prepared in long-chain fat diacid and thionyl chloride reaction, the dosage of thionyl chloride is excessive, decompression
It is distilled off having neither part nor lot in the thionyl chloride of reaction, adds dichloromethane dissolving acyl chlorides；Organic base is added dropwise to acyl chlorides and the tert-butyl alcohol
Mixture, ester of the reaction generation with cl radical, be evaporated under reduced pressure after removing solvent, add the pre- solvent for being cooled to 0~4 DEG C
The A aqueous solution, precipitation is collected by filtration；Solvent in precipitation is removed, then extracted toward addition solvent B in precipitation, weight after solvent evaporated
Crystallization, obtains the tert-butyl ester of long-chain fat diacid one；
2) tert-butyl ester of long-chain fat diacid one is dissolved in solvent C, is cooled to -20 DEG C~-10 DEG C, add N- hydroxyl ambers
Amber acid imide (HOSu) and dicyclohexylcarbodiimide (DCC), continue reaction 2 hours at -20 DEG C~-10 DEG C, recover to 15~
30 DEG C are continued reaction 3~16 hours, are filtered to remove precipitation, are evaporated under reduced pressure and are removed solvent, recrystallization, with solvent D filter wash cakes, obtain
Long-chain fat diacid succinimido tertiary butyl ester；
3) long-chain fat diacid succinimido tertiary butyl ester and the Pidolidone -1- tert-butyl esters are dissolved in N, N- diformazans
In base formamide, it is stirred overnight under the conditions of existing for organic base, is evaporated under reduced pressure and removes solvent, residue is dissolved in ethyl acetate,
And washed successively with hydrochloric acid solution, water and saturated nacl aqueous solution, anhydrous sodium sulfate drying filtering, filtrate decompression is distilled, obtained
The oily residue arrived, it is tert-butyl group long-chain fat diacyl-L-Glu-OtBu；
4) tert-butyl group long-chain fat diacyl-L-Glu-OtBu is dissolved in solvent E, is cooled to -20 DEG C~-10 DEG C,
N-hydroxysuccinimide (HOSu) and carbodicyclo hexylimide (DCC) are added, it is small to continue reaction 2 at -20 DEG C~-10 DEG C
When, recover to 15~30 DEG C to react 3~16 hours, be filtered to remove precipitation, be evaporated under reduced pressure and remove solvent, it is straight into residual grease
Connect and add solvent F precipitation solids, precipitation is collected by filtration, obtains tert-butyl group long-chain fat diacyl-L-Glu (OSu)-OtBu；
5) tert-butyl group long-chain fat diacyl-L-Glu (OSu)-OtBu is dissolved in trifluoroacetic acid, 15~30 DEG C of stirrings
Reaction, into reaction solution, addition solvent G directly separates out solid, collected by suction solid, obtains long-chain fat diacid derivative.
For solvent orange 2 A-G used for dissolving or purification is used, its dosage and access times are unlimited in above-mentioned reaction.
But consider from cost and the effect reached, respectively define solvent orange 2 A-G dosage below.
The condition that long-chain fat diacid and thionyl chloride reaction are prepared to acyl chlorides described in step 1) adds for 80 DEG C
Heat flows back 3 hours or stayed overnight under DMF catalysis in 15~30 DEG C of reactions.
Described is preferably as follows under DMF catalysis in 15~30 DEG C of overnight operations of reaction：Long-chain fat diacid is resuspended
In dichloromethane, DMF and thionyl chloride are subsequently added into, in 15~30 DEG C of reactions overnight.
Organic base described in step 1) is in monoethyl amine, diethylamine, triethylamine, diisopropylethylamine, pyridine and piperidines
One kind or at least two；Preferably pyridine.
The mole dosage of organic base described in step 1) equivalent to described long-chain fat diacid mole 0.6~
1.5 again；Preferably 1.0~1.2 times；More preferably 1.04~1.05 times.
The addition time control of organic base described in step 1) within 2 hours, otherwise occur in course of reaction compared with
More impurity, difficulty is brought to subsequent purification.
The operating condition that organic base is added dropwise to the mixture of acyl chlorides and the tert-butyl alcohol described in step 1) is as follows：Reaction temperature
For 20~30 DEG C, preferably 20~25 DEG C；Reaction time is 2~16 hours, preferably 2~4 hours, more preferably 3 hours.
Solvent orange 2 A described in step 1) be preferably it is a kind of in N,N-dimethylformamide, acetonitrile, acetone and tetrahydrofuran or
At least two, preferably DMF.
The volume ratio of solvent orange 2 A and water is 1 in the solvent orange 2 A aqueous solution described in step 1)：1~5, preferably 1：2~3, more
Preferably 1：2.
The solvent orange 2 A aqueous solution described in step 1) must be cooled to 0~4 DEG C in advance when adding, and this is advantageous to control operation mistake
The impurity generated in journey.
The dosage of the solvent orange 2 A aqueous solution described in step 1) is excessive；It is preferred that press its volume matter with long-chain fat diacid
It is 10~50mL to measure ratio：1g is calculated；Preferably press 20~30mL：1g is calculated；More preferably press 30mL：1g is calculated.
The solvent removal in precipitation being removed preferably by constant weight is dried under vacuum to described in step 1).
The number of extraction described in step 1) is preferably at least 2 times, to increase purity.
Solvent B described in step 1) is one kind or at least two in dichloromethane, ethyl acetate, petroleum ether and normal heptane
Kind, preferably petroleum ether.
The dosage that solvent B described in step 1) is extracted every time by the volume mass ratio of itself and long-chain fat diacid for 10~
50mL：1g is calculated, preferably by 10~20mL：1g is calculated.
The number of described extraction is 2 times.
In the operation of recrystallization described in step 1), used solvent is one kind or two in petroleum ether and normal heptane
Kind, preferably normal heptane.It is 4mL that the dosage of solvent, which presses itself and the volume mass ratio of long-chain fat diacid, in described recrystallization：1g
.
Solvent C described in step 2) is preferably one kind or at least two in tetrahydrofuran, ethyl acetate and dichloromethane
Kind；Preferably dichloromethane.
The dosage of described solvent C is 10 with the described tert-butyl ester volume mass ratio of long-chain fat diacid one by it~
50mL：Even if 1g；Preferably 19~21mL：1g is calculated.
The dosage of dicyclohexylcarbodiimide described in step 2) presses itself and the described tert-butyl ester of long-chain fat diacid one
Mol ratio is 1~1.2：1, preferably 1.1：1.
The temperature of cooling described in step 2) is preferably -20 DEG C~-15 DEG C, more preferably -20 DEG C.
Recovery to 15~30 DEG C of times for continuing reaction described in step 2) are preferably 12~16 hours.
Solvent used is preferably one kind in methanol, ethanol and isopropanol or at least in recrystallization described in step 2)
Two kinds, preferably isopropanol.
Solvent D described in step 2) is preferably one or both of petroleum ether and normal heptane；Preferably normal heptane.
Described solvent D dosage preferably by its with the described tert-butyl ester volume mass of long-chain fat diacid one than 1~
5mL：1g is calculated, more preferably by 3~5mL：1g is calculated.
The dosage of N,N-dimethylformamide described in step 3) is sub- with described long-chain fat diacid succinyl by it
Amido tertiary butyl ester volume mass ratio is 10~50mL：1g is calculated, preferably by 19~21mL：1g is calculated.
Organic base described in step 3) is preferably monoethyl amine, diethylamine, triethylamine, DIPEA, pyridine
With one kind in piperidines or at least two；Preferably N, N- diisopropylethylamine.
The dosage of described organic base presses itself and described long-chain fat diacid succinimido tertiary butyl ester mol ratio
For 1~2：1 calculates；It is preferred that by 1.2~1.5：1 calculates；More preferably press 1.5：1 calculates.
The dosage of ethyl acetate described in step 3) presses itself and the described tertiary fourth of long-chain fat diacid succinimido
Base ester volume mass ratio is 10~50mL：1g is calculated, preferably by 19~21mL：1g is calculated.
The concentration of hydrochloric acid solution described in step 3) is 0.1~0.2M, preferably 0.2M.
Solvent E described in step 4) is preferably one kind or at least two in tetrahydrofuran, ethyl acetate and dichloromethane
Kind, preferably dichloromethane.
Described solvent E dosage presses itself and described tert-butyl group long-chain fat diacyl-L-Glu-OtBu volume mass
Than for 10~50mL：1g is calculated；Preferably press 19~21mL：1g is calculated.
The temperature of cooling described in step 4) is preferably -20 DEG C~-15 DEG C, more preferably -20 DEG C.
Recovery to 15~30 DEG C of times for continuing reaction described in step 4) are preferably 12~16 hours.
The dosage of dicyclohexylcarbodiimide described in step 4) by its with described tert-butyl group long-chain fat diacyl-
L-Glu-OtBu mol ratios are 1~1.2：1 calculates, preferably by 1.1：1 calculates.
Solvent F described in step 4) is preferably one or both of petroleum ether and normal heptane；It is preferred that normal heptane.
Described solvent F dosage preferably presses itself and described tert-butyl group long-chain fat diacyl-L-Glu-OtBu volumes
5~20mL of mass ratio：1g is calculated, more preferably by 8~10mL：1g is calculated.
The dosage of trifluoroacetic acid described in step 5) presses itself and described tert-butyl group long-chain fat diacyl-L-Glu
(OSu)-OtBu volume mass ratio is 3~10mL: 1g calculating；Preferably calculated by 3~5mL: 1g；More preferably based on 5mL: 1g
Calculate.
The temperature of stirring reaction described in step 5) is preferably 15~30 DEG C, preferably 25 DEG C.
The time of stirring reaction described in step 5) is preferably 1~3 hour；More preferably 2~3 hours.
Solvent G described in step 5) is preferably at least two in tetrahydrofuran, absolute ether, petroleum ether and normal heptane
The mixed solvent of formation；The solvent that preferably absolute ether and normal heptane are mixed to get；More preferably absolute ether and normal heptane
1: 2~5 solvent being mixed to get by volume；Most preferably absolute ether and normal heptane 1: 3~4 are mixed to get by volume
Solvent.
Described solvent G dosage preferably presses itself and described butyl long-chain fat diacyl-L-Glu (OSu)-OtBu bodies
10~50mL of product mass ratio：1g is calculated, more preferably by 9~11mL：1g is calculated.
The method for preparing fat diacid derivative is particularly suitable for preparing insulin analog.
The present invention is had the following advantages relative to prior art and effect：
(1) route of preparation method provided by the invention is easy to operate, quality controllable, is adapted to industry's enlarging production.
(2) acyl chlorides is employed in first step synthetic intermediate D of the present invention technique and tert-butyl alcohol reaction replaces N, N- diformazans
Base formamide di-t-butyl acetal, has saved cost, Simultaneous purification is simple.
(3) n-hydroxysuccinimide/dicyclohexyl carbon is employed in second step synthetic intermediate E of the present invention technique
Diimine replaces 2- succinimidos -1,1,3,3- tetramethylurea tetrafluoro boric acid esters (TSTU), same economically feasible.
(4) the 5th step of the invention takes off t-butyl ester product H subsequent operation simple possibles, and product quality is controllable, can directly lead to
Cross the insulin analog that protein modified technology is used to prepare high-purity.
Embodiment
With reference to embodiment, the present invention is described in further detail, but the implementation of the present invention is not limited to this.
Embodiment 1
First, the preparation of the tert-butyl ester of hexadecandioic acid (hexadecane diacid) one
Hexadecandioic acid (hexadecane diacid) (20.0g, 69.8mmol) is suspended in thionyl chloride (100ml, 1376.8mmol), 80 DEG C of heating
Backflow 3 hours, it is evaporated under reduced pressure and removes excessive thionyl chloride.Dichloromethane (200ml) dissolving is added, adds the tert-butyl alcohol
(7.34ml, 76.81mmol) and pyridine (5.9ml, 73.32mmol), pyridine add in 2 hours, 25 DEG C of reaction 3h.Evaporated under reduced pressure
Solvent, add pre- DMF/ water mixed solvents 600ml (DMF and the water by volume 1 for being cooled to 0~4 DEG C：2 proportioning mixing), filtering is received
Collection precipitation.Constant weight is dried under vacuum to, dichloromethane (300ml) is added and is resuspended, collect evaporated under reduced pressure solvent after filtrate, add oil
Ether (400ml) be resuspended, collect filtrate decompression be evaporated, add normal heptane (80ml) recrystallization, precipitation is collected by filtration, be dried under reduced pressure to
Constant weight, the tert-butyl ester of hexadecandioic acid (hexadecane diacid) one is obtained, yield 10.32g, yield 40%, HPLC detection purity is 99.4%.
ESI-MS m/z：342.3[M+H]+, meet with theoretical value.
2nd, the preparation of hexadecandioic acid (hexadecane diacid) succinimido tertiary butyl ester
The tert-butyl ester of hexadecandioic acid (hexadecane diacid) one (10.3g, 30.1mmol) is dissolved in dichloromethane (200ml), under the conditions of -20 DEG C
N-hydroxysuccinimide (3.64g, 31.6mmol) and dicyclohexylcarbodiimide (6.84g, 33.1mmol) are separately added into ,-
20 DEG C are continued reaction 2 hours, are recovered room temperature (25 DEG C) and are stirred overnight.Filter, evaporated under reduced pressure solvent, add isopropanol (90ml) weight
Crystallization, is collected by filtration filter cake, and filter cake is washed with normal heptane (30ml), and it (is hexadecandioic acid (hexadecane diacid) succinimido to collect filter cake
Tertiary butyl ester), it is dried under reduced pressure to constant weight, weight 10.2g, yield：77%, HPLC purity：99.4%.
ESI-MS m/z：439.3[M+H]+, meet with theoretical value.
3rd, tert-butyl hexadecandioyl diacyl-L-Glu-OtBu preparation
Hexadecandioic acid (hexadecane diacid) succinimido tertiary butyl ester (10.18g, 23.2mmol) is taken to be dissolved in N, N- dimethyl formyls
Amine (200ml), sequentially add Pidolidone α-tert-butyl ester (5.18g, 25.5mmol), DIPEA (5.94ml,
34.7mmol), it is stirred overnight at room temperature.Evaporated under reduced pressure solvent DMF and residual DIPEA, it is residual
Remaining grease ethyl acetate (200ml) dissolves.0.2M HCl, purified water and saturated common salt water washing, anhydrous slufuric acid are used successively
Sodium filters after drying, and is sticky oil thing (being tert-butyl hexadecandioyl diacyl-L-Glu-OtBu), weight after evaporated under reduced pressure solvent
Measure 12.3g, yield：100%, HPLC purity：98.8%.
ESI-MS m/z：527.4[M+H]+, meet with theoretical value.
4th, tert-butyl hexadecandioyl diacyl-L-Glu (OSu)-OtBu preparation
Tert-butyl hexadecandioyl diacyl-L-Glu-OtBu (11.63g, 22.04mmol) is dissolved in dichloromethane under ice bath
(220ml).N-hydroxysuccinimide (2.66g, 24.14mmol) is separately added under the conditions of -20 DEG C and dicyclohexyl carbon two is sub-
Amine (5.0g, 24.24mmol), -20 DEG C are continued reaction 2 hours, are recovered to being stirred overnight at room temperature.Filter, evaporated under reduced pressure solvent obtains
To grease.Normal heptane (100ml) is added, solid precipitation is collected by filtration, is dried under reduced pressure to constant weight, obtains tert-butyl hexadecandioyl two
Acyl group-L-Glu (OSu)-OtBu, weight 13.22g, yield：96%, HPLC purity：99.2%.
ESI-MS m/z：624.4[M+H]+, meet with theoretical value.
5th, hexadecane diacyl-L-Glu (OSu) preparation
Tert-butyl hexadecandioyl diacyl-L-Glu (OSu)-OtBu (13.2g, 21.12mmol) is dissolved in trifluoroacetic acid
(66ml).It is stirred at room temperature 2 hours, absolute ether (130ml) and normal heptane (390ml) is successively added under condition of ice bath.Filter and receive
Collect solid precipitation, be dried under reduced pressure to constant weight, obtain hexadecane diacyl-L-Glu (OSu), weight 9.96g, yield：92%,
HPLC purity：92.7%.
ESI-MS m/z：512.3[M+H]+, meet with theoretical value.
6th, application of the fat diacid derivative in the preparation of moral paddy insulin
Des (B30) actrapid monotards prepare that (Zhuhai federation pharmacy share is limited according to patent CN 103290083B embodiments 1
Company produces, lot number P1231404007).Des (B30) actrapid monotards 10g is dissolved in 160ml water, ice bath stirring, treats temperature
When degree is down to 3 DEG C, triethylamine is added to adjust pH11.8.According to hexadecane diacyl-L-Glu (OSu): Des (B30) actrapid monotard mole
Than weighing hexadecane diacyl-L-Glu (OSu) 1.08g for 1.2: 1,8.75ml NMP (METHYLPYRROLIDONE) are dissolved in
In.
Hexadecane diacyl-the L-Glu (OSu) of dissolving is slowly homogeneously added into the Des of ice bath stirring with peristaltic pump
(B30) it is stirring while adding in people's pancreas islet, added in 30-35min.(w/v) % of 0.327ml 40 methylamine water solutions are added to terminate instead
Should.
HPLC analysis shows form 62% moral paddy insulin：LysB29 (N ε-hexadecane diacyl-gamma-glutamyl
Base) (B30) actrapid monotard is taken off, the moral paddy insulin that purity is higher than 99.7% can be prepared after purification.
ESI-MS：m/z：1221.7[M+5H]5+, 1526.8 [M+4H]4+, 1018.0 [M+6H]6+, meet with theoretical value.
Embodiment 2
First, the preparation of the tert-butyl ester of tetracosandioic acid one
Tetracosandioic acid (20.0g, 77.4mmol) is suspended in dichloromethane (200ml), is separately added into catalysis equivalent
DMF (89.8 μ l, 1.16mmol), thionyl chloride (33.73ml, 464.4mmol), 25 DEG C are stirred overnight, until dissolving.Decompression is steamed
Solvent is removed in distillation, adds dichloromethane (200ml) dissolving, add the tert-butyl alcohol (8.07ml, 84.49mmol) and pyridine (6.5ml,
80.65mmol), pyridine adds in 2 hours, 20 DEG C of reaction 3h.Evaporated under reduced pressure solvent, add the pre- DMF/ water for being cooled to 0~4 DEG C and mix
Bonding solvent 600ml (DMF and water by volume 1：3 proportioning mixing), precipitation is collected by filtration.Constant weight is dried under vacuum to, adds dichloro
Methane (300ml) is resuspended, and collects evaporated under reduced pressure solvent after filtrate, adds petroleum ether (400ml) and is resuspended, and collects filtrate decompression and steams
It is dry, normal heptane (80ml) recrystallization is added, precipitation is collected by filtration, is dried under reduced pressure to constant weight, obtains the tert-butyl ester of tetracosandioic acid one,
Yield 10.22g, yield：42%, HPLC purity：99.1%.
ESI-MS m/z：314.2[M+H]+, meet with theoretical value.
2nd, the preparation of tetracosandioic acid succinimido tertiary butyl ester
The tert-butyl ester of tetracosandioic acid one (10.2g, 32.4mmol) is dissolved in dichloromethane (200ml), under the conditions of -20 DEG C
N-hydroxysuccinimide (3.92g, 34.1mmol) and dicyclohexylcarbodiimide (7.37g, 35.67mmol) are separately added into ,-
20 DEG C are continued reaction 2 hours, are recovered 15 DEG C and are stirred overnight.Filter, evaporated under reduced pressure solvent, add isopropanol (90ml) recrystallization,
Filter cake is collected by filtration, washs filter cake with normal heptane (30ml), collects filter cake, be dried under reduced pressure to constant weight, obtain tetracosandioic acid amber
Amber imide tertiary butyl ester, weight 10.67g, yield：80%, HPLC purity：99.1%.
ESI-MS m/z：411.3[M+H]+, meet with theoretical value.
3rd, tert-butyl group tetradecane diacyl-L-Glu-OtBu preparation
Tetracosandioic acid succinimido tertiary butyl ester (10.6g, 25.76mmol) is taken to be dissolved in N, N- dimethyl formyls
Amine (200ml), sequentially add Pidolidone α-tert-butyl ester (5.79g, 28.49mmol), DIPEA (6.64ml,
38.77mmol), it is stirred overnight at room temperature.Evaporated under reduced pressure DMF and residual DIPEA, residual oil
Shape thing ethyl acetate (200ml) dissolves.Done successively with 0.2M HCl, purified water and saturated common salt water washing, anhydrous sodium sulfate
Filtered after dry, be sticky oil thing after evaporated under reduced pressure solvent, weight 12.87g, yield：100%, HPLC purity：98.5%.
ESI-MS m/z：499.4[M+H]+, meet with theoretical value.
4th, tert-butyl group tetradecane diacyl-L-Glu (OSu)-OtBu preparation
Tert-butyl group tetradecane diacyl-L-Glu-OtBu (12.8g, 25.62mmol) is dissolved in dichloromethane under ice bath
(250ml).N-hydroxysuccinimide (3.09g, 28.06mmol) is separately added under the conditions of -20 DEG C and dicyclohexyl carbon two is sub-
Amine (5.81g, 28.17mmol), -20 DEG C are continued reaction 2 hours, recover to 15 DEG C to be stirred overnight.Filter, evaporated under reduced pressure solvent obtains
To grease.Normal heptane (100ml) is added, solid precipitation is collected by filtration, is dried under reduced pressure to constant weight, weight 14.07g, yield：
92%, HPLC purity：98.9%.
ESI-MS m/z：596.4[M+H]+, meet with theoretical value.
5th, tetradecane diacyl-L-Glu (OSu) preparation
Tert-butyl group tetradecane diacyl-L-Glu (OSu)-OtBu (14g, 23.46mmol) is dissolved in trifluoroacetic acid
(70ml).It is stirred at room temperature 2 hours, absolute ether (140ml) and normal heptane (420ml) is successively added under condition of ice bath.Filter and receive
Collect solid precipitation, be dried under reduced pressure to constant weight, weight 10.57g, yield：93%, HPLC purity：92.5%.
ESI-MS m/z：484.2[M+H]+, meet with theoretical value.
6th, application of the fat diacid derivative in the preparation of insulin analog
Des (B30) actrapid monotards prepare according to patent CN 103290083B embodiments 1, by Des (B30) actrapid monotard
10g is dissolved in 160ml water, ice bath stirring, when temperature is down to 3 DEG C, adds triethylamine to adjust pH11.8.According to the acyl of the tetradecane two
Base-L-Glu (OSu): Des (B30) actrapid monotard mol ratio weighs tetradecane diacyl-L-Glu (OSu) 1.02g for 1.2: 1,
It is dissolved in 8.75ml NMP (METHYLPYRROLIDONE).
Tetradecane diacyl-the L-Glu (OSu) of dissolving is slowly homogeneously added into the Des of ice bath stirring with peristaltic pump
(B30) it is stirring while adding in people's pancreas islet, added in 30-35min.Add the methylamine water solution terminating reactions of 0.31ml 40%.
HPLC analysis shows form 63% insulin analog：LysB29 (N ε-tetradecane diacyl-gamma-glutamyl
Base) (B30) actrapid monotard is taken off, the insulin analog that purity is higher than 99.7% can be prepared after purification.
ESI-MS：m/z：1216.1[M+5H]5+, 1519.8 [M+4H]4+, 1013.3 [M+6H]6+, meet with theoretical value.
Embodiment 3
First, the preparation of the tert-butyl ester of octadecane diacid one
Octadecane diacid (20.0g, 63.6mmol) is suspended in dichloromethane (200ml), is separately added into catalysis equivalent
DMF (73.8 μ l, 0.95mmol), thionyl chloride (27.72ml, 381.6mmol), are stirred overnight at room temperature, until dissolving.Decompression is steamed
Solvent is removed in distillation, adds dichloromethane (200ml) and dissolves, the addition tert-butyl alcohol (6.63ml, 69.43mmol), pyridine (5.34ml,
66.27mmol), pyridine adds in 2 hours, 25 DEG C of reaction 3h.Evaporated under reduced pressure solvent, add the pre- DMF/ water for being cooled to 0~4 DEG C and mix
Bonding solvent 600ml (DMF and water by volume 1：2 proportioning mixing), precipitation is collected by filtration.Constant weight is dried under vacuum to, adds dichloro
Methane (200ml) is resuspended, and collects evaporated under reduced pressure solvent after filtrate, adds petroleum ether (400ml) and is resuspended, and collects filtrate decompression and steams
It is dry, normal heptane (80ml) recrystallization is added, precipitation is collected by filtration, is dried under reduced pressure to constant weight, yield 9.19g, yield：39%,
HPLC purity：99.3%.
ESI-MS m/z：370.3[M+H]+, meet with theoretical value.
2nd, the preparation of octadecane diacid succinimido tertiary butyl ester
The tert-butyl ester of octadecane diacid one (9.1g, 24.56mmol) is being dissolved in dichloromethane (180ml), -20 DEG C of conditions
Under be separately added into n-hydroxysuccinimide (2.97g, 25.85mmol) and dicyclohexylcarbodiimide (5.59g,
27.04mmol), -20 DEG C are continued reaction 2 hours, are recovered 30 DEG C and are stirred overnight.Filter, evaporated under reduced pressure solvent, add isopropanol
(80ml) is recrystallized, and filter cake is collected by filtration, and is washed filter cake with normal heptane (30ml), is collected filter cake, be dried under reduced pressure to constant weight, weight
9.3g, yield：81%, HPLC purity：99.3%.
ESI-MS m/z：476.3[M+H]+, meet with theoretical value.
3rd, tert-butyl group octadecandioyl-L-Glu-OtBu preparation
Octadecane diacid succinimido tertiary butyl ester (9.3g, 19.89mmol) is taken to be dissolved in N, N- dimethyl formyls
Amine (180ml), sequentially add Pidolidone α-tert-butyl ester (4.47g, 21.99mmol), DIPEA (5.13ml,
29.93mmol), it is stirred overnight at room temperature.Evaporated under reduced pressure DMF and residual DIPEA, residual oil
Shape thing ethyl acetate (180ml) dissolves.Done successively with 0.2M HCl, purified water and saturated common salt water washing, anhydrous sodium sulfate
Filtered after dry, be sticky oil thing after evaporated under reduced pressure solvent, weight 11.07g, yield：100%, HPLC purity：98.5%.
ESI-MS m/z：555.4[M+H]+, meet with theoretical value.
4th, tert-butyl group octadecandioyl-L-Glu (OSu)-OtBu preparation
Tert-butyl group octadecandioyl-L-Glu-OtBu (11.0g, 19.76mmol) is dissolved in dichloromethane under ice bath
(220ml).N-hydroxysuccinimide (2.38g, 21.64mmol) is separately added under the conditions of -20 DEG C and dicyclohexyl carbon two is sub-
Amine (4.48g, 21.72mmol), -20 DEG C are continued reaction 2 hours, recover to 30 DEG C to be stirred overnight.Filter, evaporated under reduced pressure solvent obtains
To grease.Normal heptane (100ml) is added, solid precipitation is collected by filtration, is dried under reduced pressure to constant weight, weight 11.5g, yield：
89%, HPLC purity：98.9%.
ESI-MS m/z：652.4[M+H]+, meet with theoretical value.
5th, octadecandioyl-L-Glu (OSu) preparation
Tert-butyl group octadecandioyl-L-Glu (OSu)-OtBu (11.5g, 17.59mmol) is dissolved in trifluoroacetic acid
(57.5ml).It is stirred at room temperature 2 hours, absolute ether (115ml) and normal heptane (345ml) is successively added under condition of ice bath.Filter
Solid precipitation is collected, is dried under reduced pressure to constant weight, weight 8.57g, yield：90%, HPLC purity：92.4%.
ESI-MS m/z：540.3[M+H]+, meet with theoretical value.
6th, application of the fat diacid derivative in the preparation of insulin analog
Des (B30) actrapid monotards prepare according to the B embodiments 1 of patent CN 103290083, by Des (B30) actrapid monotard
10g is dissolved in 160ml water, ice bath stirring, when temperature is down to 3 DEG C, adds triethylamine to adjust pH11.8.According to the acyl of octadecane two
Base-L-Glu (OSu): Des (B30) actrapid monotard mol ratio weighs octadecandioyl-L-Glu (OSu) 1.42g for 1.5: 1,
It is dissolved in 8.75ml NMP (METHYLPYRROLIDONE).
Octadecandioyl-the L-Glu (OSu) of dissolving is slowly homogeneously added into the Des of ice bath stirring with peristaltic pump
(B30) it is stirring while adding in people's pancreas islet, added in 30-35min.Add the methylamine water solution terminating reactions of 0.406ml 40%.
HPLC analysis shows form 71% insulin analog：LysB29 (N ε-octadecandioyl-gamma-glutamyl
Base) (B30) actrapid monotard is taken off, the insulin analog that purity is higher than 99.7% can be prepared after purification.
ESI-MS：m/z：1227.3[M+5H]5+, 1533.8 [M+4H]4+, 1022.7 [M+6H]6+, meet with theoretical value.
Embodiment 4
First, the preparation of the tert-butyl ester of eicosane diacid one
Eicosane diacid (20.0g, 58.39mmol) is suspended in thionyl chloride (100ml), 80 DEG C are heated to reflux 3 hours,
It is evaporated under reduced pressure and removes excessive thionyl chloride.Dichloromethane (200ml) is added to dissolve, the addition tert-butyl alcohol (6.14ml,
64.26mmol), pyridine (4.94ml, 61.34mmol), pyridine add in 2 hours, 20 DEG C of reaction 3h.Evaporated under reduced pressure solvent, adds
Enter pre- DMF/ water mixed solvents 600ml (DMF and the water by volume 1 for being cooled to 0~4 DEG C：2 proportioning mixing), precipitation is collected by filtration.
Constant weight is dried under vacuum to, the dichloromethane (200ml) that addition is pre-chilled to 0~4 DEG C is resuspended, and collects evaporated under reduced pressure solvent after filtrate,
Add petroleum ether (400ml) to be resuspended, collect filtrate decompression and be evaporated, add normal heptane (80ml) recrystallization, precipitation is collected by filtration, subtracts
Press dry dry to constant weight, yield 8.85g, yield：38%, HPLC purity：99.4%.
ESI-MS m/z：398.3[M+H]+, meet with theoretical value.
2nd, the preparation of eicosane diacid succinimido tertiary butyl ester
The tert-butyl ester of eicosane diacid one (8.8g, 22.1mmol) is being dissolved in dichloromethane (180ml), -20 DEG C of conditions
Under be separately added into n-hydroxysuccinimide (2.67g, 23.2mmol) and dicyclohexylcarbodiimide (5.02g,
24.3mmol), -20 DEG C are continued reaction 2 hours, are recovered 25 DEG C and are stirred overnight.Filter, evaporated under reduced pressure solvent, add isopropanol
(79ml) is recrystallized, and filter cake is collected by filtration, and is washed filter cake with normal heptane (30ml), is collected filter cake, be dried under reduced pressure to constant weight, weight
8.75g yield：80%, HPLC purity：99.4%.
ESI-MS m/z：495.4[M+H]+, meet with theoretical value.
3rd, tert-butyl group eicosane diacyl-L-Glu-OtBu preparation
Eicosane diacid succinimido tertiary butyl ester (8.7g, 17.55mmol) is taken to be dissolved in N, N- dimethyl formyls
Amine (180ml), sequentially add Pidolidone α-tert-butyl ester (3.92g, 19.29mmol), DIPEA (4.49ml,
26.25mmol), it is stirred overnight at room temperature.Evaporated under reduced pressure DMF and residual DIPEA, residual oil
Shape thing ethyl acetate (180ml) dissolves.Done successively with 0.2M HCl, purified water and saturated common salt water washing, anhydrous sodium sulfate
Filtered after dry, be sticky oil thing after evaporated under reduced pressure solvent, weight 10.25g, yield：100%, HPLC purity：98.6%.
ESI-MS m/z：583.4[M+H]+, meet with theoretical value.
4th, tert-butyl group eicosane diacyl-L-Glu (OSu)-OtBu preparation
Tert-butyl group eicosane diacyl-L-Glu-OtBu (10.2g, 17.47mmol) is dissolved in dichloromethane under ice bath
(200ml).N-hydroxysuccinimide (2.11g, 19.14mmol) is separately added under the conditions of -20 DEG C and dicyclohexyl carbon two is sub-
Amine (3.96g, 19.21mmol), -20 DEG C are continued reaction 2 hours, recover to 25 DEG C to be stirred overnight.Filter, evaporated under reduced pressure solvent obtains
To grease.Normal heptane (100ml) is added, solid precipitation is collected by filtration, is dried under reduced pressure to constant weight, weight 11.18g, yield：
94%, HPLC purity：99.2%.
ESI-MS m/z：679.5[M+H]+, meet with theoretical value.
5th, eicosane diacyl-L-Glu (OSu) preparation
Tert-butyl group eicosane diacyl-L-Glu (OSu)-OtBu (11.1g, 16.3mmol) is dissolved in trifluoroacetic acid
(55.5ml).It is stirred at room temperature 2 hours, absolute ether (110ml) and normal heptane (330ml) is successively added under condition of ice bath.Filter
Solid precipitation is collected, is dried under reduced pressure to constant weight, weight 8.44g, yield：91%, HPLC purity：92.2%.
ESI-MS m/z：567.3[M+H]+, meet with theoretical value.
6th, application of the fat diacid derivative in the preparation of moral paddy insulin
Des (B30) actrapid monotards prepare according to patent CN 103290083B embodiments 1, by Des (B30) actrapid monotard
10g is dissolved in 160ml water, ice bath stirring, when temperature is down to 3 DEG C, adds triethylamine to adjust pH11.8.According to the acyl of eicosane two
Base-L-Glu (OSu): Des (B30) actrapid monotard mol ratio weighs eicosane diacyl-L-Glu (OSu) 1.49g for 1.5: 1,
It is dissolved in 8.75ml NMP (METHYLPYRROLIDONE).
Eicosane diacyl-the L-Glu (OSu) of dissolving is slowly homogeneously added into the Des of ice bath stirring with peristaltic pump
(B30) it is stirring while adding in people's pancreas islet, added in 30-35min.Add the methylamine water solution terminating reactions of 0.407ml 40%.
HPLC analysis shows form 69% insulin analog：LysB29 (N ε-eicosane diacyl-gamma-glutamyl
Base) (B30) actrapid monotard is taken off, the insulin analog that purity is higher than 99.7% can be prepared after purification.
ESI-MS：m/z：1232.9[M+5H]5+, 1540.9 [M+4H]4+, 1027.3 [M+6H]6+, meet with theoretical value.
Comparative example 1
Work is prepared according to the tert-butyl ester of hexadecandioic acid (hexadecane diacid) one disclosed in Novo Nordisk Co., Ltd's patent No. CN1829738 embodiments 4
Skill, hexadecandioic acid (hexadecane diacid) (20.0g, 69.8mmol) is suspended in toluene (125ml), and the mixture is heated to flowing back.It is small 4
When it is interior dropwise addition DMF di-t-butyl acetal (38.2g, 187.5mmol), by the mixture flow back overnight.50℃
Remove solvent in lower vacuum, be suspended in DCM/AcOEt (250ml, 1: in 1), stir 15 minutes.Solid is collected by filtration, adds DCM
(50ml) is resuspended, and filtrate is collected by filtration, and is evaporated under reduced pressure, and obtained product is suspended in the pre- DCM (25ml) for being cooled to 0 DEG C, temperature drop
To 4 DEG C of filterings, it is evaporated under reduced pressure and removes solvent, adds in normal heptane (100ml) and recrystallize, obtain the tert-butyl ester of hexadecandioic acid (hexadecane diacid) one
7.9g (33%), be not suitable for industry amplification.
Comparative example 2
According to hexadecandioic acid (hexadecane diacid) succinimido uncle disclosed in Novo Nordisk Co., Ltd's patent No. CN1829738 embodiments 4
Butyl ester preparation technology, the tert-butyl ester of hexadecandioic acid (hexadecane diacid) one (2g, 5.8mmol) is dissolved in tetrahydrofuran (20ml), adds TSTU
(2.1g, 7.0mmol) and DIPEA (1.2ml, 7.0mmol), is stirred overnight at room temperature.Filtrate is collected by filtration, subtracts
Solvent is distilled off in pressure, and residue is dissolved in into ethyl acetate, with precooling 0.1M HCl and water washing twice, is done with anhydrous sodium sulfate
Dry filtering, it is evaporated under reduced pressure and removes solvent, obtain hexadecandioic acid (hexadecane diacid) succinimido tertiary butyl ester 2.0g (79%).The technique into
This height, be not suitable for industry amplification.
Comparative example 3
According to prepared disclosed in Grinstaff and Zhang the tert-butyl ester of dodecanedioic acid one technique (Grinstaff and
Zhang et al., Bioconjugate chemistry, 2011,22 (4), 690-9) tert-butyl ester of hexadecandioic acid (hexadecane diacid) one is prepared, by ten
Six docosandioic acids (18.6g, 65mmol) condition of ice bath low suspension adds the tert-butyl alcohol (64ml, 650mmol) in tetrahydrofuran (99ml)
With DMAP (0.4g, 3.25mmol), temperature is down to 2 DEG C, adds two hexamethylenes for being dissolved in tetrahydrofuran (25ml)
Base carbodiimide (16g, 78mmol), 2 DEG C are continued stirring 2 hours, are recovered room temperature and are continued reaction 24 hours.LCMS detections are main raw
Into impurity hexadecandioic acid (hexadecane diacid) N- acylureas, impurity E SI-MS：m/z：492.4[M+H]+, theoretical value：492.73 and purpose is produced
The tert-butyl ester ratio of thing hexadecandioic acid (hexadecane diacid) one is very low, is not suitable for industry amplification.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (6)
- A kind of 1. method for preparing fat diacid derivative, it is characterised in that comprise the following steps：(1) the long-chain fat diacid as shown in formula A and thionyl chloride are reacted, obtains carrying two cl radicals as shown in formula B Acyl chlorides；Then acyl chlorides and the tert-butyl alcohol are reacted, obtains the ester with a cl radical as shown in formula C；Then taken with hydroxyl For cl radical, the tert-butyl ester of long-chain fat diacid one as shown in formula D is obtained；Wherein, the m in following reaction equations is 6~32 Integer；(2) tert-butyl ester of long-chain fat diacid one and n-hydroxysuccinimide are reacted, obtains the long-chain fat two as shown in formula E Sour succinimido tertiary butyl ester；(3) long-chain fat diacid succinimido tertiary butyl ester and the Pidolidone -1- tert-butyl esters are reacted, obtained such as formula F institutes The tert-butyl group long-chain fat diacyl-L-Glu-OtBu shown；(4) tert-butyl group long-chain fat diacyl-L-Glu-OtBu and n-hydroxysuccinimide are reacted, obtained as shown in formula G Tert-butyl group long-chain fat diacyl-L-Glu (OSu)-OtBu；(5) tert-butyl group in tert-butyl group long-chain fat diacyl-L-Glu (OSu)-OtBu is removed, obtains the length as shown in formula H Chain fatty diacid derivative；Comprise the following steps that：1) acyl chlorides is prepared in long-chain fat diacid and thionyl chloride reaction, the dosage of thionyl chloride is excessive, is evaporated under reduced pressure The thionyl chloride for having neither part nor lot in reaction is removed, adds dichloromethane dissolving acyl chlorides；Organic base is added dropwise to the mixed of acyl chlorides and the tert-butyl alcohol Compound, ester of the reaction generation with a cl radical, is evaporated under reduced pressure after removing solvent, adds the pre- solvent orange 2 A water for being cooled to 0~4 DEG C Solution, precipitation is collected by filtration；Solvent in precipitation is removed, then toward addition solvent B extractions in precipitation, is evaporated weight after partial solvent Crystallization, obtains the tert-butyl ester of long-chain fat diacid one；2) tert-butyl ester of long-chain fat diacid one is dissolved in solvent C, is cooled to -20 DEG C~-10 DEG C, add N- hydroxysuccinimidyl acyls Imines and dicyclohexylcarbodiimide, continue reaction 2 hours at -20 DEG C~-10 DEG C, recover to 15~30 DEG C continue reaction 3~ 16 hours, precipitation is filtered to remove, is evaporated under reduced pressure and removes solvent, recrystallization, with solvent D filter wash cakes, obtain long-chain fat diacid amber Amber imide tertiary butyl ester；3) long-chain fat diacid succinimido tertiary butyl ester and the Pidolidone -1- tert-butyl esters are dissolved in N, N- dimethyl methyls In acid amides, it is stirred overnight under the conditions of existing for organic base, is evaporated under reduced pressure and removes solvent, residue is dissolved in ethyl acetate, and according to It is secondary to be washed with hydrochloric acid solution, water and saturated nacl aqueous solution, anhydrous sodium sulfate drying filtering, filtrate decompression is distilled, obtained Oily residue, it is tert-butyl group long-chain fat diacyl-L-Glu-OtBu；4) tert-butyl group long-chain fat diacyl-L-Glu-OtBu is dissolved in solvent E, is cooled to -20 DEG C~-10 DEG C, added N-hydroxysuccinimide and carbodicyclo hexylimide, continue reaction 2 hours at -20 DEG C~-10 DEG C, recover to 15~30 DEG C Reaction 3~16 hours, precipitation is filtered to remove, is evaporated under reduced pressure and removes partial solvent, solvent F analysis is directly added into residual grease Go out solid, precipitation is collected by filtration, obtain tert-butyl group long-chain fat diacyl-L-Glu (OSu)-OtBu；5) tert-butyl group long-chain fat diacyl-L-Glu (OSu)-OtBu is dissolved in trifluoroacetic acid, 15~30 DEG C of stirrings are anti- Should, into reaction solution, addition solvent G directly separates out solid, collected by suction solid, obtains long-chain fat diacid derivative；Solvent orange 2 A described in step 1) is one kind or at least two in N,N-dimethylformamide, acetonitrile, acetone and tetrahydrofuran Kind；Solvent B described in step 1) is one kind or at least two in dichloromethane, ethyl acetate, petroleum ether and normal heptane；Organic base described in step 1) is one in monoethyl amine, diethylamine, triethylamine, diisopropylethylamine, pyridine and piperidines Kind or at least two；Used solvent is one or two kinds of in petroleum ether and normal heptane in recrystallization described in step 1)；Solvent C described in step 2) is one kind or at least two in tetrahydrofuran, ethyl acetate and dichloromethane；Solvent used is one kind or at least two in methanol, ethanol and isopropanol in recrystallization described in step 2)；Solvent D described in step 2) is one or both of petroleum ether and normal heptane；Organic base described in step 3) is monoethyl amine, in diethylamine, triethylamine, DIPEA, pyridine and piperidines One kind or at least two；Solvent E described in step 4) is one kind or at least two in tetrahydrofuran, ethyl acetate and dichloromethane；Solvent F described in step 4) is one or both of petroleum ether and normal heptane；Solvent G described in step 5) is the mixed of at least two formation in tetrahydrofuran, absolute ether, petroleum ether and normal heptane Bonding solvent.
- 2. the method according to claim 1 for preparing fat diacid derivative, it is characterised in that：It is 6~23.6 that the dosage of thionyl chloride described in step 1), which presses itself and the mol ratio of described long-chain fat diacid,：1 meter Calculate；The dosage of the tert-butyl alcohol described in step 1) is 0.6~1.5 by the tert-butyl alcohol and long-chain fat diacid mole ratio：1 calculates；The dosage of n-hydroxysuccinimide described in step 2) presses itself and the described tert-butyl ester mole of long-chain fat diacid one Than for 1~1.2：1 calculates；The dosage of the Pidolidone -1- tert-butyl esters described in step 3) presses itself and described long-chain fat diacid succinimido Tertiary butyl ester mol ratio is 1~1.5：1 calculates；The dosage of n-hydroxysuccinimide described in step 4) presses itself and described tert-butyl group long-chain fat diacyl-L- Glu-OtBu mol ratios are 1~1.2：1 calculates.
- 3. the method according to claim 2 for preparing fat diacid derivative, it is characterised in that：The dosage of the tert-butyl alcohol described in step 1) is 1~1.2 by the tert-butyl alcohol and long-chain fat diacid mole ratio：1 calculates；The dosage of n-hydroxysuccinimide described in step 2) presses itself and the described tert-butyl ester mole of long-chain fat diacid one Than for 1.05：1 calculates；The dosage of the Pidolidone -1- tert-butyl esters described in step 3) presses itself and described long-chain fat diacid succinimido Tertiary butyl ester mol ratio is 1~1.2：1 calculates；The dosage of n-hydroxysuccinimide described in step 4) presses itself and described tert-butyl group long-chain fat diacyl-L- Glu-OtBu mol ratios are 1.1：1 calculates.
- 4. the method according to claim 1 for preparing fat diacid derivative, it is characterised in that：The condition that long-chain fat diacid and thionyl chloride reaction are prepared to acyl chlorides described in step 1) heats back for 80 DEG C Flow 3 hours or stayed overnight under DMF catalysis in 15~30 DEG C of reactions；The temperature of cooling described in step 2) is -20 DEG C~-15 DEG C；Recovery to 15~30 DEG C of times for continuing reaction described in step 2) are 12~16 hours；The concentration of hydrochloric acid solution described in step 3) is 0.1~0.2M；The temperature of cooling described in step 4) is -20 DEG C~-15 DEG C；Recovery to 15~30 DEG C of times for continuing reaction described in step 4) are 12~16 hours；The time of stirring reaction described in step 5) is 1~3 hour.
- 5. the method according to claim 1 for preparing fat diacid derivative, it is characterised in that：0.6~1.5 times of the mole dosage of organic base described in step 1) equivalent to described long-chain fat diacid mole；It is 10~50mL that the dosage of the solvent orange 2 A aqueous solution described in step 1), which presses itself and the volume mass ratio of long-chain fat diacid,： 1g is calculated；The dosage that solvent B described in step 1) is extracted every time by the volume mass ratio of itself and long-chain fat diacid for 10~ 50mL：1g is calculated；The dosage of solvent C described in step 2) is 10 with the described tert-butyl ester volume mass ratio of long-chain fat diacid one by it~ 50mL：1g is calculated；The dosage of dicyclohexylcarbodiimide described in step 2) presses itself and the described tert-butyl ester mole of long-chain fat diacid one Than for 1~1.2：1 calculates；The dosage of solvent D described in step 2) by its with the described tert-butyl ester volume mass of long-chain fat diacid one than 1~ 5mL：1g is calculated；The dosage of N,N-dimethylformamide described in step 3) presses itself and described long-chain fat diacid succinimido Tertiary butyl ester volume mass ratio is 10~50mL：1g is calculated；The dosage of organic base described in step 3) is rubbed by itself and described long-chain fat diacid succinimido tertiary butyl ester You are than being 1~2：1 calculates；The dosage of ethyl acetate described in step 3) presses itself and described long-chain fat diacid succinimido tertiary butyl ester Volume mass ratio is 10~50mL：1g is calculated；The dosage of solvent E described in step 4) presses itself and described tert-butyl group long-chain fat diacyl-L-Glu-OtBu volumes Mass ratio is 10~50mL：1g is calculated；The dosage of dicyclohexylcarbodiimide described in step 4) presses itself and described tert-butyl group long-chain fat diacyl-L- Glu-OtBu mol ratios are 1~1.2：1 calculates；The dosage of solvent F described in step 4) presses itself and described tert-butyl group long-chain fat diacyl-L-Glu-OtBu volumes 5~20mL of mass ratio：1g is calculated；The dosage of trifluoroacetic acid described in step 5) by its with described tert-butyl group long-chain fat diacyl-L-Glu (OSu)- OtBu volume mass ratio is 3~10mL: 1g calculating；The dosage of solvent G described in step 5) presses itself and described butyl long-chain fat diacyl-L-Glu (OSu)-OtBu bodies 10~50mL of product mass ratio：1g is calculated.
- 6. any one of Claims 1 to 5 method for preparing fat diacid derivative answering in insulin analog is prepared With.
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