CN103335873B - Method for measuring chloride ion content of amino acid-N-formic anhydride - Google Patents
Method for measuring chloride ion content of amino acid-N-formic anhydride Download PDFInfo
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Abstract
The invention relates to and prepares a solution which can be used for measuring the chloride ion content of N-carboxylic anhydride, and relates to a method for measuring the chloride ion content of N-carboxylic anhydride. The invention further relates to a method for preparing high-purity N-carboxylic anhydride and prepared high-purity N-carboxylic anhydride. In addition, the invention further relates to a method for preparing high-purity polypeptide mixture and prepared high-purity polypeptide mixture which is glatiramer acetate especially.
Description
Technical field
The present invention relates to prepare can be used for the solution for measuring chloride ion content in N- carboxy acid anhydrides, and measurement N- carboxylic acids
The method of chloride ion content in acid anhydride.The invention further relates to prepare the method with high purity N-carboxy acid anhydride, and prepare
With high purity N-carboxy acid anhydride.Moreover, it relates to prepare the method with high-purity mixtures of polypeptides, Yi Jiyou
This mixtures of polypeptides for preparing, in particular, mixtures of polypeptides is acetic acid copaxone.
Background technology
Amino acid N-carboxy acid anhydride（I.e., hereinafter designated as " N- carboxy acid anhydrides " or " amino acid N CA "）It is 3- Qu Dai Evil
Oxazoline -2,5- diketone are as follows：
Due to from α-, β-, the N- carboxy acid anhydrides that obtain in gamma-amino acid（It is abbreviated as NCA）Acid functional group work
Change can cause which to become the compound with reactivity.This is because N- carboxy acid anhydrides cause the acidic functional group with it is any
Nucleophilic body is reacted to possibility.Therefore so that prepared by carrying out amide functional group with amine functions radical reaction
Simplify.Thus, N- carboxy acid anhydrides are generally used for synthesis polypeptide.Additionally, N- carboxy acid anhydrides easily form ester bond with alcohol reactant liquor.
Have several known methods can be used for prepare N- carboxy acid anhydrides, such as US6656458, US4267344 and
US6479665.Most common and most straightforward approach is in solvent by aminoacid or its hydrochlorate with phosgene, surpalite or triphosgene
React in medium.
It is as shown below with the reaction of phosgene：
Wherein, R represent α-, β-, the main free radical of gamma-amino acid, R ' represents the secondary amino group of hydrogen atom or aminoacid
Free radical, R ' can form ring with R.
, it is known that a large amount of hydrogen chloride gas are yet forms both in addition to N- carboxy acid anhydrides during synthesis N- carboxy acid anhydrides, that is,
Say per mole of N- carboxy acid anhydrides production 2mol hydrogen chloride gas.Hydrogen chloride gas are that with high response, in media as well, hydrogen chloride gas are deposited
In the appearance that would potentially result in side reaction and chlorinated secondary product.In terms of quality and yield, the chlorine in N- carboxy acid anhydrides is remained in
It is absolutely not desired to change impurity.Presence due to remaining in hydrogen chloride gas in N- carboxy acid anhydrides, can severe jamming N- carboxy acid anhydrides
Polyreaction.Can be smoothed out to be polymerized, need the chloride ion content present in N- carboxy acid anhydride monomers sufficiently low.Cause
This, can the content of free chlorine usually need to be less than 0.05wt%.
Very eurypalynous mixtures of polypeptides, such as acetic acid copaxone is gathered by the N- carboxy acid anhydrides of aminoacid
Close and prepare.For example, acetic acid copaxone (GA, also referred to as copolymer-1, Cop-1 or, CAS
No.147245-92-9), it passes through four kinds of aminoacid（L-Alanine, L-Tyrosine, L-Glutamic Acid-γ-benzyl ester, L- ε-trifluoro
Acetyl-lysine）N- carboxy acid anhydrides（NCA）The mixtures of polypeptides for being polymerized at random and being prepared, with following structural formula：
(Glu,Ala,Lys,Tyr)xXCH3COOH
(C5H9NO4·C3H7MO2·C6H14N202·C9H11NO3)x'XC2H4O2
Acetic acid copaxone is a kind of artificial synthetic polypeptide mixture for treating multiple sclerosis
（M.M.Mouradain,Pharmacology&Therapeutics,98,245-255,2003）.Specifically refer to
Physician ' s Desk Reference, Thomson PDR, Montvale, New Jersey, page 3297 (2007).Vinegar
The amino acid molar ratio of sour copaxone is about 0.392-0.462:0.129-0.153:0.300-0.374:0.086-
0.100, mean molecule quantity about 5000-9000 dalton.
Inventor's discovery, prepares mixtures of polypeptides by N- carboxy acid anhydrides（Such as, acetic acid copaxone）When, N- carboxyls
The quality of anhydride, especially content of dissociative chlorine ion are very big for the qualitative effects of polyreaction and product.Especially, if
N- carboxy acid anhydrides can not reach should specification, and the mean molecule quantity of acetic acid copaxone and aminoacid composition can not all be reached and should be had
Scope：The amino acid molar ratio of acetic acid copaxone is about 0.392-0.462:0.129-0.153:0.300-0.374:
0.086-0.100, mean molecule quantity about 5000-9000 dalton.
In order that the mixtures of polypeptides for preparing（Such as, acetic acid copaxone）Product can be reached to require accordingly, the present invention
Inventor's discovery, preferred purity >=99.5% of N- carboxy acid anhydrides, chloride ion<0.01%, free aminoacid content<0.01%.
As the chloride ion content in N- carboxy acid anhydrides is a kind of key factor, which affects amino acid N-carboxy acid anhydride matter
Amount, and then affect mixtures of polypeptides quality.But, N- carboxy acid anhydrides are highly unstable to water and alcohols, can aggregate into and not dissolve in
The polymer of water.And as the water solublity of polymer is excessively poor, directly affects the accuracy to chloride ion detection by quantitative.Ability
Domain needs the detection method efficiently and accurately for obtaining the chloride ion content with regard to N- carboxy acid anhydrides and can be used to measure N- carboxylics
The solution of chloride ion content in base anhydride.
The content of the invention
One purpose of the present invention is to provide and can be used for the solution for measuring chloride ion content in N- carboxy acid anhydrides, and measurement
The method of chloride ion content in N- carboxy acid anhydrides.
Another object of the present invention is to provide the method with high purity N-carboxy acid anhydride for preparing, and the tool for preparing
There is high purity N-carboxy acid anhydride.
Additionally, still another object of the present invention is to provide to be related to prepare the method with high-purity mixtures of polypeptides, and
Thus the mixtures of polypeptides for preparing, in particular, mixtures of polypeptides is acetic acid copaxone.
The present invention can be used for the solution for measuring chloride ion content in N- carboxy acid anhydrides, its method system by comprising the steps
It is standby：
1. N- carboxy acid anhydrides are dissolved in one or more solvent, wherein the solvent is substantially anhydrous solvent,
2. the N- carboxy acid anhydrides in solvent are dissolved in using hydrolysing agent hydrolysis, obtain can be used to measure chlorine in N- carboxy acid anhydrides
The solution of ion concentration.
The method of chloride ion content in present invention measurement N- carboxy acid anhydrides, which comprises the steps：
1. prepare and can be used for the solution for measuring chloride ion content in N- carboxy acid anhydrides, which includes：
（a）N- carboxy acid anhydrides are dissolved in one or more solvent, wherein the solvent is substantially anhydrous solvent,
（b）The N- carboxy acid anhydrides in solvent are dissolved in using hydrolysing agent hydrolysis, obtain can be used to measure in N- carboxy acid anhydrides
The solution of chloride ion content.
2. chloride ion content in N- carboxy acid anhydrides obtained above is measured.
The present invention prepares the method with high purity N-carboxy acid anhydride, and which includes conventional use known to those skilled in the art
Preparation with high purity N-carboxy acid anhydride method, which further includes following step：
- chloride ion content in the N- carboxy acid anhydrides for obtaining is detected as follows；
(1). preparation can be used for the solution for measuring chloride ion content in N- carboxy acid anhydrides, and which includes：
（a）N- carboxy acid anhydrides are dissolved in one or more solvent,
（b）The N- carboxy acid anhydrides in solvent are dissolved in using hydrolysing agent hydrolysis, obtain can be used to measure in N- carboxy acid anhydrides
The solution of chloride ion content.
(2). chloride ion content in measurement N- carboxy acid anhydrides obtained above；
When chloride ion content is more than 0.05wt%, preferably greater than 0.01wt%, then next step purification step is carried out.
In said method, solvent is substantially anhydrous solvent, and does not include alcohol, for example：
- solvent miscible with water, especially selected from dimethyl sulfoxide (DMSO), DMF (DMF), N, N-
Dimethyl acetylamide (DMA), -2 ketopyrrolidine of N- methyl (NMP), dimethyl sulfoxide (DMSO), Methanamide and sulfolane, dioxy
Six rings, tetrahydrofuran (THF), acetone, acetonitrile and carboxylic acid such as formic acid, acetic acid etc.；
The liquid salt of-ionic liquid, such as Alkylated imidazole.
By the above-mentioned invention scope that the present invention is fallen within high purity N-carboxy acid anhydride for preparing.
Additionally, the present invention is provided prepares the method with high-purity mixtures of polypeptides, which includes conventional people in the art
The method for preparing mixtures of polypeptides used known to member, which further includes following step：
Chloride ion content in-following detection raw material N- carboxy acid anhydrides, when chloride ion content is more than 0.05wt%, preferably greater than
During 0.01wt%, then next step purification step is carried out；
(1). preparation can be used for the solution for measuring chloride ion content in N- carboxy acid anhydrides, and which includes：
（a）N- carboxy acid anhydrides are dissolved in one or more solvent,
（b）The N- carboxy acid anhydrides in solvent are dissolved in using hydrolysing agent hydrolysis, obtain can be used to measure in N- carboxy acid anhydrides
The solution of chloride ion content.
（2）Chloride ion content in detection raw material N- carboxy acid anhydrides.
Solvent is substantially anhydrous solvent in the above-mentioned methods, and does not include alcohol, for example：
- solvent miscible with water, especially selected from dimethyl sulfoxide (DMSO), DMF (DMF), N, N-
Dimethyl acetylamide (DMA), -2 ketopyrrolidine of N- methyl (NMP), dimethyl sulfoxide (DMSO), Methanamide and sulfolane, dioxy
Six rings, tetrahydrofuran (THF), acetone, acetonitrile and carboxylic acid such as formic acid, acetic acid etc.；
The liquid salt of-ionic liquid, such as Alkylated imidazole.
(2). chloride ion content in measurement N- carboxy acid anhydrides obtained above.
The invention further relates to the mixtures of polypeptides for thus preparing, in particular, acetic acid copaxone falls within the present invention
Invention scope.In order that the mixtures of polypeptides for preparing（Such as, acetic acid copaxone）Product can be reached to require accordingly, this
Bright inventor's discovery, preferred purity >=99.5% of raw material N- carboxy acid anhydrides, chloride ion<0.01%, free aminoacid content<0.01%.
Purification step used in the present invention can use conventional method well known in the art, such as recrystallization, activated carbon
Absorption, by methods of purification such as packed columns.
The method that can be used for the measurement chloride ion content of the present invention is a lot, and the methods known to those skilled in the art can be with
Use in the present invention, for example chromatography such as column chromatography, paper chromatography, thin layer chromatography etc.；Silver nitrate turbidimetry method；Point
Light photometry；Mole method-Neutral potassium chromate makees indicator；Method is raised the administration of justice-fluorescein and makees indicator；Fo Erjiadefa-indirect titration；Electricity
Position titrimetry etc..In the above-mentioned methods, it is preferable that under inert gas shielding, by N- carboxy acid anhydrides be dissolved in one or more it is molten
In agent.Noble gases can be nitrogen, argon, helium, neon, argon, krypton, xenon, radon etc..
In the present invention, the water yield that substantially anhydrous solvent contains in referring to solvent is less than 10wt%, particularly less than 5wt%, excellent
Choosing is less than 3%, preferably smaller than 1%.
In the present invention, using can be so that the material of N- carboxy acid anhydrides hydrolysis be hydrolyzed.Hydrolysing agent is used for example
As water or aqueous alkali are hydrolyzed, preferred water or sodium hydrate aqueous solution.Wherein, water is not comprising impact chloride ion measurement
Water, the i.e. not water of chloride ion-containing, preferred deionized water.
In the present invention, during hydrolysis is dissolved in the N- carboxy acid anhydrides in solvent, hydrolysing agent such as water or aqueous alkali
Usage amount be：0.1-50mL/mg N- carboxy acid anhydrides, especially preferred 1-30mL/mg N- carboxy acid anhydrides, 1-20mL/mg N- carboxylics
Base anhydride, more preferably 1-10mL/mg N- carboxy acid anhydrides.
According to the inventive method, N- carboxy acid anhydrides can be broken to the aqueous solution of good water solubility, then using various skills
Art accurately measures the chloride ion content of N- carboxy acid anhydrides.The method of the chloride ion content of accurate detection N- carboxy acid anhydride of the present invention
Chloride test result will not be caused to be forbidden because of polymer wrapped problem is produced.
For the purposes of the present invention, state " aminoacid " to be understood to refer to comprising at least one NR1R2 groups and extremely
Any compound of a few carboxylic group, wherein R1 and R2 is any group that can form aminoacid, for example hydrogen, alkyl,
Aryl, or R1 and R2 formation cyclic groups.The aminoacid of the present invention can be naturally occurring or synthetic source.In addition to glycine, day
So aminoacid all contains chiral carbon atom.All it is possible to using natural or non-natural aminoacid.Aminoacid can have D
Or L-configuration.
In the present invention, N- carboxy acid anhydrides are, for example, sarcosine N- carboxy acid anhydrides, threonine N- carboxy acid anhydrides, serine N-
Carboxy acid anhydride, L-Valine N- carboxy acid anhydride, norvaline N- carboxy acid anhydrides, isoleucine N- carboxy acid anhydrides, leucine N- carboxyls
Anhydride, nor-leucine N- carboxy acid anhydrides, lysine N- carboxy acid anhydrides, Phenylalanine N- carboxy acid anhydride, L-Tyrosine N- carboxy acid anhydride,
Alanine N- carboxy acid anhydrides, proline N- carboxy acid anhydrides, tryptophan N- carboxy acid anhydrides, methionine N- carboxy acid anhydrides, glycine N-
Carboxy acid anhydride, serine N- carboxy acid anhydrides, cysteine N- carboxy acid anhydrides, arginine N- carboxy acid anhydrides, histidine N- carboxylic acids
Acid anhydride, tryptophan N- carboxy acid anhydrides, methionine N- carboxy acid anhydrides, aspartic acid N- carboxy acid anhydrides and glutamic acid N-carboxy acid anhydride,
Glutamic acid-γ-benzyl ester N- carboxy acid anhydride, ε-trifluoroacetyl group-lysine N- carboxy acid anhydrides etc..
The residue of the aminoacid that can be used is according to following 3 alphabetical code abbreviations：Alanine (Ala), arginine
(Arg), aspartic acid (Asp), agedoite (Asn), cysteine (Cys), glutamic acid (Glu), glutamine (Gln), sweet
Propylhomoserin (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), benzene
Alanine (Phe), serine (Ser), threonine (Thr), tryptophan (Trp), L-Tyrosine (Tyr) and L-Valine (Val), have
The aminoacid of nucleophilic side-chains is advantageously protected by side chain using them in the method according to the invention before.
Statement " protection group " is understood to refer to any kind of group, and it prevents coupled atom or group (example
Such as oxygen atom or nitrogen-atoms) undesirable reaction is participated in building-up process.These protection groups include Side chain protective group and
The group of protection C- or N- ends, is referred to as amine protecting group and sour protection group.
As the example of the exhaustive of amine protecting group, it can be mentioned that particularly benzoyl (Bz), acetyl group (Ac), trifluoro
Acetyl group (Tfa), benzyloxycarbonyl group (Z), to benzyloxycarbonylchloride base (2ClZ), to bromo-benzyloxycarbonyl (2BrZ), to nitrobenzyloxycarbonyl,
Hexichol methbxybenzyl-oxycarbonyl, hexichol methoxycarbonyl group, 9-fluorenylmethyloxycarbonyl (Fmoc), tertbutyloxycarbonyl (Boc), benzenesulfonyl,
P-toluenesulfonyl or 2- nitrobenzenesulfonyls.
As the example of the exhaustive of sour protection group, it can be mentioned that alkyl, aryl, aralkyl, silyl type, such as first
Epoxide methyl, methylthiomethyl, 2,2,2- trichloroethyls, 2- halogenated ethyls, 2- (TMS)-ethyl, the tert-butyl group,
Aryl, alkyl, aralkyl, pi-allyl, phenyl, trityl (trityl), benzhydryl, to nitrobenzyl, to methoxybenzyl
And the group of trialkylsilkl, such as trimethyl silicane ethers, triethylsilyl, t-butyldimethylsilyl,
Or i-propyldimethylsilyl.
For the purposes of the present invention, term " peptide " refers to a kind of polymer, and the monomer in the polymer is by amide-type
The aminoacid that the covalent bond of type links together.Peptide includes at least two aminoacid.Preferably, the number of the aminoacid in peptide chain
More than or equal to 3.Peptide chain warp often includes most 100 aminoacid.Preferably, in peptide chain, the number of aminoacid is less equal than
20.It is particularly preferred that the number of aminoacid is less equal than 15 in peptide chain.
For the purposes of the present invention, term " polypeptide " refers to the compound that a-amino acid is formed so that peptide chain links together,
It is also the intermediate product of protein hydrolysis.Dipeptides is called by the compound of two amino acid molecular dehydrating condensations, in the same manner
Analogize also tripeptides, tetrapeptide, pentapeptide etc..Generally by the compound of 10-100 amino acid molecular dehydrating condensations polypeptide.
For the purposes of the present invention, term " high purity N-carboxy acid anhydride " refers to N- carboxy acid anhydride purity >=99.5%, chlorine from
Son<0.01%, free aminoacid content<0.01%.
For the purposes of the present invention, term " high-purity mixtures of polypeptides " is referred to by raw material high purity N-carboxy acid anhydride（It is pure
Degree >=99.5%, chloride ion<0.01%, free aminoacid content<0.01%）The mixtures of polypeptides for preparing.
In the present invention, abbreviation " NCA " represents amino acid, n-carboxyanhydrides.
The invention further relates to following technical proposals：
1. the method for measuring chloride ion content in N- carboxy acid anhydrides, which comprises the steps：
（1）. preparation can be used for the solution for measuring chloride ion content in N- carboxy acid anhydrides, and which includes：
（a）N- carboxy acid anhydrides are dissolved in one or more substantially anhydrous solvent,
（b）The N- carboxy acid anhydrides in solvent are dissolved in using hydrolysing agent hydrolysis,
（c）Obtain can be used for the solution for measuring chloride ion content in N- carboxy acid anhydrides,
（2）. chloride ion content in measurement N- carboxy acid anhydrides obtained above.
2. the method according to technical scheme 1, wherein substantially anhydrous solvent is selected from as follows：
- solvent miscible with water, wherein, solvent is preferably selected from dimethyl sulfoxide (DMSO), DMF
(DMF), N,N-dimethylacetamide (DMA), -2 ketopyrrolidine of N- methyl (NMP), dimethyl sulfoxide (DMSO), Methanamide and ring
Fourth sulfone, dioxane, tetrahydrofuran (THF), acetone, acetonitrile and carboxylic acid such as formic acid, acetic acid；And/or
The liquid salt of-ionic liquid, such as Alkylated imidazole.
3. the method according to any one of above-mentioned technical proposal, wherein, under inert gas shielding, N- carboxy acid anhydrides are dissolved in
In one or more substantially anhydrous solvent.
4. the method according to any one of above-mentioned technical proposal, wherein, enter water-filling using hydrolysing agent such as water or aqueous alkali
Solution, preferred water or sodium hydrate aqueous solution, in particular, deionized water.
5. the method according to any one of above-mentioned technical proposal, wherein, hydrolysis is dissolved in the process of the N- carboxy acid anhydrides in solvent
In, the usage amount of hydrolysing agent such as water or aqueous alkali is 0.1-10mL/mgN- carboxy acid anhydrides.
6. can be used for measuring the solution of chloride ion content in N- carboxy acid anhydrides, which is prepared by the method for comprising the steps：
（a）N- carboxy acid anhydrides are dissolved in one or more substantially anhydrous solvent,
（b）The N- carboxy acid anhydrides in solvent are dissolved in using hydrolysing agent hydrolysis.
7. the method according to technical scheme 6, wherein substantially anhydrous solvent is selected from as follows：
- solvent miscible with water, wherein, solvent is preferably selected from dimethyl sulfoxide (DMSO), DMF
(DMF), N,N-dimethylacetamide (DMA), -2 ketopyrrolidine of N- methyl (NMP), dimethyl sulfoxide (DMSO), Methanamide and ring
Fourth sulfone, dioxane, tetrahydrofuran (THF), acetone, acetonitrile and carboxylic acid such as formic acid, acetic acid；And/or
The liquid salt of-ionic liquid, such as Alkylated imidazole.
8. the method according to any one of above-mentioned technical proposal 6-7, wherein, under inert gas shielding, by N- carboxy acid anhydrides
It is dissolved in one or more substantially anhydrous solvent.
9. the method according to any one of above-mentioned technical proposal 6-8, wherein, carried out using hydrolysing agent such as water or aqueous alkali
Hydrolysis, preferred water or sodium hydrate aqueous solution, in particular, deionized water.
10. the method according to any one of above-mentioned technical proposal 6-9, wherein, hydrolysis is dissolved in the N- carboxy acid anhydrides in solvent
During, the usage amount of hydrolysing agent such as water or aqueous alkali is 0.1-10mL/mgN- carboxy acid anhydrides.
11. methods for preparing N- carboxy acid anhydrides, which comprises the steps：
A. N- carboxy acid anhydrides, wherein chloride ion content is selected to be more than 0.05wt%, preferably greater than 0.01wt% as follows：
Chloride ion content in the N- carboxy acid anhydrides that-detection is obtained；
(1). preparation can be used for the solution for measuring chloride ion content in N- carboxy acid anhydrides, and which includes：
（a）N- carboxy acid anhydrides are dissolved in one or more solvent,
（b）The N- carboxy acid anhydrides in solvent are dissolved in using hydrolysing agent hydrolysis, obtain can be used to measure in N- carboxy acid anhydrides
The solution of chloride ion content；
(2). chloride ion content in measurement N- carboxy acid anhydrides obtained above；
When chloride ion content is more than 0.05wt%, preferably greater than 0.01wt%, then carries out purification step and cause its chloride ion
Content is less than 0.05wt%, preferably smaller than 0.01wt%.
12. according to the method for technical scheme 11, wherein substantially anhydrous solvent is selected from as follows：
- solvent miscible with water, wherein, solvent is preferably selected from dimethyl sulfoxide (DMSO), DMF
(DMF), N,N-dimethylacetamide (DMA), -2 ketopyrrolidine of N- methyl (NMP), dimethyl sulfoxide (DMSO), Methanamide and ring
Fourth sulfone, dioxane, tetrahydrofuran (THF), acetone, acetonitrile and carboxylic acid such as formic acid, acetic acid；And/or
The liquid salt of-ionic liquid, such as Alkylated imidazole.
13. according to the method for any one of above-mentioned technical proposal 11-12, wherein, under inert gas shielding, by N- carboxylic acids
Acid anhydride is dissolved in one or more substantially anhydrous solvent.
14. according to the method for any one of above-mentioned technical proposal 11-13, wherein, using hydrolysing agent such as water or aqueous alkali
It is hydrolyzed, preferred water or sodium hydrate aqueous solution, in particular, deionized water.
15. according to the method for any one of above-mentioned technical proposal 11-14, wherein, hydrolysis is dissolved in the N- carboxy acid anhydrides in solvent
During, the usage amount of the hydrolysing agent such as water or aqueous alkali is：0.1-50mL/mgN- carboxy acid anhydrides, preferred 1-
30mL/mg N- carboxy acid anhydrides, especially 1-20mL/mg N- carboxy acid anhydrides, more preferably 1-10mL/mg N- carboxy acid anhydrides.
16. methods for preparing polypeptide, which comprises the steps：
A. N- carboxy acid anhydrides, wherein chloride ion content is selected to be more than 0.05wt%, preferably greater than 0.01wt% as follows：
Chloride ion content in the N- carboxy acid anhydrides that-detection is obtained；
(1). preparation can be used for the solution for measuring chloride ion content in N- carboxy acid anhydrides, and which includes：
（a）N- carboxy acid anhydrides are dissolved in one or more solvent,
（b）The N- carboxy acid anhydrides in solvent are dissolved in using hydrolysing agent hydrolysis, obtain can be used to measure in N- carboxy acid anhydrides
The solution of chloride ion content；
(2). chloride ion content in measurement N- carboxy acid anhydrides obtained above；
When chloride ion content is more than 0.05wt%, preferably greater than 0.01wt%, then carries out purification step and cause its chloride ion
Content is less than 0.05wt%, preferably smaller than 0.01wt%.
17. according to the method for technical scheme 16, and wherein polypeptide is acetic acid copaxone.
18. according to the method for any one of technical scheme 16-17, wherein substantially anhydrous solvent is selected from as follows：
- solvent miscible with water, wherein, solvent is preferably selected from dimethyl sulfoxide (DMSO), DMF
(DMF), N,N-dimethylacetamide (DMA), -2 ketopyrrolidine of N- methyl (NMP), dimethyl sulfoxide (DMSO), Methanamide and ring
Fourth sulfone, dioxane, tetrahydrofuran (THF), acetone, acetonitrile and carboxylic acid such as formic acid, acetic acid；And/or
The liquid salt of-ionic liquid, such as Alkylated imidazole.
19. according to the method for any one of above-mentioned technical proposal 16-18, wherein, under inert gas shielding, by N- carboxylic acids
Acid anhydride is dissolved in one or more substantially anhydrous solvent.
20. according to the method for any one of above-mentioned technical proposal 16-19, wherein, using hydrolysing agent such as water or aqueous alkali
It is hydrolyzed, preferred water or sodium hydrate aqueous solution, in particular, deionized water.
21. according to the method for any one of above-mentioned technical proposal 16-20, wherein, hydrolysis is dissolved in the N- carboxy acid anhydrides in solvent
During, the usage amount of the hydrolysing agent such as water or aqueous alkali is：0.1-50mL/mgN- carboxy acid anhydrides, preferred 1-
30mL/mg N- carboxy acid anhydrides, especially 1-20mL/mg N- carboxy acid anhydrides, more preferably 1-10mL/mg N- carboxy acid anhydrides.
Following instance is intended to illustrate the present invention without limiting the present invention.
Specific embodiment
Embodiment 1. tests the preparation of L-Alanine-NCA sample solutions
About 20mg L-Alanine-NCA measuring samples are weighed accurately under nitrogen protection, with 1 milliliter of anhydrous Isosorbide-5-Nitrae-dioxy six
Ring（Water content≤0.04%）Dissolving.By the solution be added drop-wise to stirring in 10 ml deionized waters in, by the mixture spend from
Sub- water is completely transferred in 20 milliliters of volumetric flasks, deionized water constant volume.
Embodiment 2. tests the preparation of L-Tyrosine-NCA sample solutions
About 20mg L-Tyrosine-NCA measuring samples are weighed accurately under nitrogen protection, with 1 milliliter of anhydrous Isosorbide-5-Nitrae-dioxy six
Ring（Water content≤0.04%）Dissolving.The solution is added drop-wise in 10 milliliters of 0.01M sodium hydrate aqueous solutions in stirring, should
Mixture deionized water is completely transferred in 20 milliliters of volumetric flasks, deionized water constant volume.
Embodiment 3. tests the preparation of L-Glutamic Acid-γ-benzyl ester-NCA sample solutions
About 20mg L-Glutamic Acid-γ-benzyl ester-NCA measuring samples are weighed accurately under nitrogen protection, with 1 milliliter anhydrous 1,
4- dioxane（Water content≤0.04%）Dissolving.The solution is added drop-wise in 10 ml deionized waters in stirring, this is mixed
Thing deionized water is completely transferred in 20 milliliters of volumetric flasks, deionized water constant volume.
Embodiment 4. tests the preparation of L- ε-trifluoroacetyl group-lysine-NCA sample solutions
About 20mg L- ε-trifluoroacetyl group-lysine-NCA measuring samples are weighed accurately under nitrogen protection, with 1 milliliter
Anhydrous 1,4- dioxane（Water content≤0.04%）Dissolving.The solution is added drop-wise in 10 ml deionized waters in stirring, will
The mixture deionized water is completely transferred in 20 milliliters of volumetric flasks, deionized water constant volume.
The preparation of 5. blank solution of embodiment
By 1 milliliter of anhydrous 1,4- dioxane（Water content≤0.04%）It is added drop-wise in 10 ml deionized waters in stirring,
The mixture deionized water is completely transferred in 20 milliliters of volumetric flasks, deionized water constant volume.
6. sample detection of embodiment
Above-mentioned four solution for preparing are detected with chromatography of ions, chromatographic column is Dionex IonPacTMAS19,
The KOH of 4.0 × 250mm, flow velocity 1.0ml/min, 10mM carries out eluting, using electric conductivity detector detection signal, chloride ion appearance
Time is about at 10.6min.Reference substance is prepared with Sodium Chloride simultaneously, is detected with external standard method.Chloride test result such as table
1。
Table 1：Chloride test result
Embodiment 7. prepares copolymer-1（That is, acetic acid copaxone）
Isosorbide-5-Nitrae-dioxane the 200mL for adding Jing metallic sodiums to process in there-necked flask, adds L-Alanine under room temperature
NCA2.590 gram, L-Tyrosine NCA1.036 gram, L-Glutamic Acid-γ-benzyl ester NCA1.974 gram, L- ε-trifluoroacetyl groups-rely ammonia
Sour NCA4.693 gram.Stirring 30 minutes, clarifies to system, adds 33 milligrams of diethylamine.20-25 degree Celsius of lower mechanical agitation 24 is little
When.Reactant liquor is poured slowly in 400mL water, a large amount of white solids is produced, after sucking filtration, is vacuum dried to obtain 7.424 grams, yield
92.8%。
7.0 grams of above-mentioned shielded copaxone, 31% hydrogen bromide/acetic acid mixture 140mL, in 22- are added in there-necked flask
Stir 22 hours under 24 degrees Celsius.Rufous reactant liquor is poured in 400mL water, a large amount of white solids are produced, sucking filtration, vacuum are done
It is dry to obtain 4.76 grams of trifluoroacetyl group copaxone.0.76 gram of above-mentioned trifluoroacetyl group copaxone, 1M piperidines are added in there-necked flask
Aqueous solution 38mL, stirs 24 hours under room temperature.Dialysed with the filter membrane of 1KDa, the solution ice acetic acid for obtaining to pH is 5.5-
5.8, stir 1 hour.Lyophilizing obtains white powder, and mean molecule quantity is 7803.
Claims (12)
1. the method for measuring chloride ion content in N- carboxy acid anhydrides, which comprises the steps：
(1) prepare for measuring the solution of chloride ion content in N- carboxy acid anhydrides, which includes：
A () is dissolved in 20mgN- carboxy acid anhydrides in the substantially anhydrous dioxane of 1mL,
B () is dissolved in the N- carboxy acid anhydrides in solvent using hydrolysing agent hydrolysis, and deionized water constant volume is to 20mL,
C () is obtained for measuring the solution of chloride ion content in N- carboxy acid anhydrides,
(2) chloride ion content in N- carboxy acid anhydrides obtained above is detected using chromatography of ions.
2. method according to claim 1, wherein, under inert gas shielding, N- carboxy acid anhydrides are dissolved in into substantially anhydrous solvent
In.
3. method according to claim 1, wherein, the use of the reagent of hydrolysis is that water or aqueous alkali are hydrolyzed.
4. method according to claim 1, wherein, the use of the reagent of hydrolysis is water or sodium hydrate aqueous solution.
5. method according to claim 1, wherein, the use of the reagent of hydrolysis is deionized water.
6. method according to claim 1, wherein, during hydrolysis is dissolved in the N- carboxy acid anhydrides in solvent, the hydrolysis examination
Agent is 0.1-10mL/mg N- carboxy acid anhydrides for the usage amount of water or aqueous alkali.
7. it is used for measuring the solution of chloride ion content in N- carboxy acid anhydrides, which is prepared by the method for comprising the steps：
A () is dissolved in 20mgN- carboxy acid anhydrides in the substantially anhydrous dioxane of 1mL,
B () is dissolved in N- carboxy acid anhydrides in solvent using hydrolysing agent hydrolysis, and deionized water constant volume is to 20mL；
Described solution is detected for chromatography of ions.
8. solution according to claim 7, wherein, under inert gas shielding, N- carboxy acid anhydrides are dissolved in into substantially anhydrous solvent
In.
9. solution according to claim 7, wherein, the use of the reagent of hydrolysis is that water or aqueous alkali are hydrolyzed.
10. solution according to claim 7, wherein, the use of the reagent of hydrolysis is water or sodium hydrate aqueous solution.
11. solution according to claim 7, wherein, the use of the reagent of hydrolysis is deionized water.
12. solution according to claim 7, wherein, during hydrolysis is dissolved in the N- carboxy acid anhydrides in solvent, the hydrolysis examination
Agent is 0.1-10mL/mg N- carboxy acid anhydrides for the usage amount of water or aqueous alkali.
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