CN103335873A - 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 the solution that can be used for chloride ion content in measuring N-carboxy acid anhydride, and the method for chloride ion content in measuring N-carboxy acid anhydride.The invention still further relates to preparation and have the method for high purity N-carboxy acid anhydride, and prepare have high purity N-carboxy acid anhydride.In addition, the invention still further relates to preparation and have the method for high-purity mixtures of polypeptides, and the mixtures of polypeptides for preparing thus, in particular, mixtures of polypeptides is that the acetic acid lattice draw for thunder.
Background technology
Amino acid N-carboxy acid anhydride (that is, hereinafter being expressed as " N-carboxy acid anhydride " or " amino acid N CA ") is 3-substituted oxazole quinoline-2, and the 5-diketone is as follows:
Since from α-, β-, the activation of the acid functional group of the N-carboxy acid anhydride (being abbreviated as NCA) that obtains the gamma-amino acid can make it become to have reactive compound.This is because the N-carboxy acid anhydride makes the reaction of this acidic functional group and any nucleophilic body become possibility.Therefore, make and to prepare simplification by carrying out the acid amides functional group with the amine functions radical reaction.Thus, the N-carboxy acid anhydride is generally used for synthetic polypeptide.In addition, N-carboxy acid anhydride and pure reactant liquor form ester bond easily.
There are several known methods to can be used for preparing the N-carboxy acid anhydride, for example US6656458, US4267344 and US6479665.Common and the most direct method is that amino acid or its hydrochloride and phosgene, surpalite or triphosgene are reacted in solvent medium.
As shown below with the reaction of phosgene:
Wherein, R represent α-, β-, the main free radical of gamma-amino acid, the free radical of R ' expression hydrogen atom or amino acid whose secondary amino group, R ' can form ring with R.
Known, in synthetic N-carboxy acid anhydride process, except the N-carboxy acid anhydride, also formed a large amount of hydrogen chloride gas, that is to say that every mole N-carboxy acid anhydride is produced the 2mol hydrogen chloride gas.Hydrogen chloride gas has high response, and in medium, the existence of hydrogen chloride gas may cause the appearance of subsidiary reaction and chlorinated secondary product.From quality and output aspect, the chlorinated impurities that remains in the N-carboxy acid anhydride is definitely undesirable.Since remain in the existence of hydrogen chloride gas in the N-carboxy acid anhydride, can the serious polyreaction of disturbing the N-carboxy acid anhydride.For polymerization can be carried out smoothly, the chloride ion content that need exist in N-carboxy acid anhydride monomer is enough low.Therefore, but the content of free chlorine usually need be less than 0.05wt%.
The mixtures of polypeptides of a lot of types, for example the acetic acid lattice draw for thunder, are to carry out polymerization by amino acid whose N-carboxy acid anhydride to prepare.For example, the acetic acid lattice draw for thunder (GA is also referred to as copolymer-1, Cop-1 or
CAS No.147245-92-9); it carries out the mixtures of polypeptides that polymerization at random prepares by the N-carboxy acid anhydride (NCA) of four seed amino acids (L-alanine, L-tyrosine, L-glutamic acid-γ-benzyl ester, L-ε-trifluoroacetyl group-lysine), has following structural formula:
(Glu,Ala,Lys,Tyr)
xXCH
3COOH
(C
5H
9NO
4·C
3H
7MO
2·C
6H
14N
20
2·C
9H
11NO
3)x'XC
2H
4O
2
It is a kind of artificial synthetic polypeptide potpourri (M.M.Mouradain, Pharmacology﹠amp that is used for the treatment of multiple sclerosis that the acetic acid lattice draw for thunder; Therapeutics, 98,245-255,2003).Specifically see also physician ' s Desk Reference, Thomson PDR, Montvale, New Jersey, the 3297th page (2007).It approximately is 0.392-0.462:0.129-0.153:0.300-0.374:0.086-0.100 that the acetic acid lattice draw the amino acid molar ratio for thunder, the about 5000-9000 dalton of mean molecular weight.
The inventor finds, by the N-carboxy acid anhydride prepare mixtures of polypeptides (as, the acetic acid lattice draw for thunder) time, the quality of N-carboxy acid anhydride, especially free chloro ion content, very big for the quality influence of polyreaction and product.Especially, if the N-carboxy acid anhydride can not reach specification should be arranged, the acetic acid lattice draw for the mean molecular weight of thunder and amino acid composition and all can not reach due scope: it approximately is 0.392-0.462:0.129-0.153:0.300-0.374:0.086-0.100 that the acetic acid lattice draw the amino acid molar ratio for thunder, the about 5000-9000 dalton of mean molecular weight.
For the mixtures of polypeptides that makes preparation (as, the acetic acid lattice draw for thunder) can reach the corresponding requirement of product, the inventor finds, the preferred purity of N-carboxy acid anhydride 〉=99.5%, chlorion＜0.01%, free aminoacid content＜0.01%.
Because the chloride ion content in the N-carboxy acid anhydride is a kind of key factor, it influences amino acid N-carboxy acid anhydride quality, and then influences the mixtures of polypeptides quality.But the N-carboxy acid anhydride is very unstable to water and alcohols, can aggregate into water-insoluble polymer.And because the water-soluble non-constant of polymkeric substance has directly influenced the accuracy that chlorion is quantitatively detected.This area need obtain about the effective and accurate detection method of the chloride ion content of N-carboxy acid anhydride and the solution that can be used for chloride ion content in measuring N-carboxy acid anhydride.
Summary of the invention
Purpose of the present invention is, the solution that can be used for chloride ion content in measuring N-carboxy acid anhydride is provided, and the method for chloride ion content in measuring N-carboxy acid anhydride.
Another purpose of the present invention is, provides preparation to have the method for high purity N-carboxy acid anhydride, and prepare have high purity N-carboxy acid anhydride.
In addition, the present invention also has another purpose to be, provide to relate to preparing to have the method for high-purity mixtures of polypeptides, and the mixtures of polypeptides for preparing thus, in particular, mixtures of polypeptides is that the acetic acid lattice draw for thunder.
The present invention can be used for the solution of chloride ion content in measuring N-carboxy acid anhydride, and it is prepared by the method that comprises the steps:
1. the N-carboxy acid anhydride is dissolved in one or more solvents, wherein this solvent is substantially anhydrous solvent,
2. the hydrolysis of use hydrolysing agent is dissolved in the N-carboxy acid anhydride in the solvent, obtains can be used for the solution of chloride ion content in measuring N-carboxy acid anhydride.
The method of chloride ion content in measuring N-carboxy acid anhydride of the present invention, it comprises the steps:
1. preparation can be used for the solution of chloride ion content in measuring N-carboxy acid anhydride, and it comprises:
(a) the N-carboxy acid anhydride is dissolved in one or more solvents, wherein this solvent is substantially anhydrous solvent,
(b) hydrolysis of use hydrolysing agent is dissolved in the N-carboxy acid anhydride in the solvent, obtains can be used for the solution of chloride ion content in measuring N-carboxy acid anhydride.
2. measure chloride ion content in the above-mentioned N-carboxy acid anhydride that obtains.
The present invention's preparation has the method for high purity N-carboxy acid anhydride, and it preparation that comprises the known use of conventional those skilled in the art has the method for high purity N-carboxy acid anhydride, and it further comprises the steps:
Chloride ion content in the N-carboxy acid anhydride that-following detection obtains;
(1). preparation can be used for the solution of chloride ion content in measuring N-carboxy acid anhydride, and it comprises:
(a) the N-carboxy acid anhydride is dissolved in one or more solvents,
(b) hydrolysis of use hydrolysing agent is dissolved in the N-carboxy acid anhydride in the solvent, obtains can be used for the solution of chloride ion content in measuring N-carboxy acid anhydride.
(2). measure chloride ion content in the above-mentioned N-carboxy acid anhydride that obtains;
When chloride ion content greater than 0.05wt%, when being preferably greater than 0.01wt%, then carry out next step purification step.
Solvent is substantially anhydrous solvent in the said method, and does not comprise alcohol, for example:
-the solvent miscible with water, especially be selected from dimethyl sulfoxide (DMSO) (DMSO), N, dinethylformamide (DMF), N, N-dimethyl acetamide (DMA), N-methyl-2 pyrrolidone (NMP), dimethyl sulfoxide (DMSO) (DMSO), formamide and sulfolane, dioxane, tetrahydrofuran (THF), acetone, acetonitrile and carboxylic acid such as formic acid, acetic acid etc.;
-ionic liquid, for example liquid salt of alkylation imidazoles.
Also belong to invention scope of the present invention by the above-mentioned high purity N-carboxy acid anhydride that has for preparing.
In addition, the invention provides preparation and have the method for high-purity mixtures of polypeptides, it comprises the method for preparing mixtures of polypeptides of the known use of conventional those skilled in the art, and it further comprises the steps:
Chloride ion content in the-following detection raw material N-carboxy acid anhydride, when chloride ion content greater than 0.05wt%, when being preferably greater than 0.01wt%, then carry out next step purification step;
(1). preparation can be used for the solution of chloride ion content in measuring N-carboxy acid anhydride, and it comprises:
(a) the N-carboxy acid anhydride is dissolved in one or more solvents,
(b) hydrolysis of use hydrolysing agent is dissolved in the N-carboxy acid anhydride in the solvent, obtains can be used for the solution of chloride ion content in measuring N-carboxy acid anhydride.
(2) detect chloride ion content in the raw material N-carboxy acid anhydride.
Solvent is substantially anhydrous solvent in said method, and does not comprise alcohol, for example:
-the solvent miscible with water, especially be selected from dimethyl sulfoxide (DMSO) (DMSO), N, dinethylformamide (DMF), N, N-dimethyl acetamide (DMA), N-methyl-2 pyrrolidone (NMP), dimethyl sulfoxide (DMSO) (DMSO), formamide and sulfolane, dioxane, tetrahydrofuran (THF), acetone, acetonitrile and carboxylic acid such as formic acid, acetic acid etc.;
-ionic liquid, for example liquid salt of alkylation imidazoles.
(2). measure chloride ion content in the above-mentioned N-carboxy acid anhydride that obtains.
The invention still further relates to the mixtures of polypeptides for preparing thus, in particular, the acetic acid lattice draw for thunder and also belong to invention scope of the present invention.For the mixtures of polypeptides that makes preparation (as, the acetic acid lattice draw for thunder) can reach the corresponding requirement of product, the inventor finds, the preferred purity of raw material N-carboxy acid anhydride 〉=99.5%, chlorion＜0.01%, free aminoacid content＜0.01%.
Employed purification step can use conventional method well known in the art among the present invention, as recrystallization, charcoal absorption, by methods of purification such as packed columns.
The method that can be used for measurement chloride ion content of the present invention is a lot, and the method known to those skilled in the art all can be used in the present invention, for example for example column chromatography, paper chromatography, thin-layered chromatography etc. of chromatography; Silver nitrate turbidimetry method; Spectrophotometric method; Mole method-potassium chromate is made indicator; Method is raised the administration of justice-fluorescein and is made indicator; Fo Erjiadefa-indirect titration; Potentiometric titration etc.In said method, preferred, under inert gas shielding, the N-carboxy acid anhydride is dissolved in one or more solvents.Inert gas can be nitrogen, argon, helium, neon, argon, krypton, xenon, radon etc.
In the present invention, the water yield that substantially anhydrous solvent refers to contain in the solvent is less than 10wt%, especially less than 5wt%, preferably less than 3%, preferably less than 1%.
In the present invention, use can be so that the material of N-carboxy acid anhydride hydrolysis be hydrolyzed.For example use hydrolysing agent such as water or aqueous alkali to be hydrolyzed, preferred water or sodium hydrate aqueous solution.Wherein, water does not comprise influences the water that chlorion is measured, i.e. the water of chloride ion-containing not, preferred deionized water.
In the present invention, hydrolysis is dissolved in the process of the N-carboxy acid anhydride in the solvent, the use amount of hydrolysing agent such as water or aqueous alkali is: 0.1-50mL/mg N-carboxy acid anhydride, preferred 1-30mL/mg N-carboxy acid anhydride, especially 1-20mL/mg N-carboxy acid anhydride, more preferably 1-10mL/mg N-carboxy acid anhydride.
According to the inventive method, the N-carboxy acid anhydride can be broken to the aqueous solution of good water solubility, utilize the accurately chloride ion content of measuring N-carboxy acid anhydride of various technology then.The method that the present invention accurately detects the chloride ion content of N-carboxy acid anhydride can not cause the chlorion testing result inaccurate because of producing polymkeric substance parcel problem.
For the purposes of the present invention, statement " amino acid " is interpreted as referring to comprise any compound of at least one NR1R2 group and at least one carboxylic group, wherein R1 and R2 anyly can form amino acid whose group, for example hydrogen, alkyl, aryl, or R1 and R2 form cyclic group.Amino acid of the present invention can be natural or synthetic source.Except glycocoll, natural amino acid all contains asymmetric carbon atom.It all is possible using natural or non-natural amino acid.Amino acid can have D or L configuration.
Among the present invention; the N-carboxy acid anhydride for example is methyl amimoacetic acid N-carboxy acid anhydride; threonine N-carboxy acid anhydride; serine N-carboxy acid anhydride; valine N-carboxy acid anhydride; norvaline N-carboxy acid anhydride; isoleucine N-carboxy acid anhydride; leucine N-carboxy acid anhydride; nor-leucine N-carboxy acid anhydride; lysine N-carboxy acid anhydride; phenylalanine N-carboxy acid anhydride; tyrosine N-carboxy acid anhydride; alanine N-carboxy acid anhydride; proline N-carboxy acid anhydride; tryptophane N-carboxy acid anhydride; methionine N-carboxy acid anhydride; glycocoll N-carboxy acid anhydride; serine N-carboxy acid anhydride; halfcystine N-carboxy acid anhydride; arginine N-carboxy acid anhydride; histidine N-carboxy acid anhydride; tryptophane N-carboxy acid anhydride; methionine N-carboxy acid anhydride; aspartic acid N-carboxy acid anhydride and glutamic acid N-carboxy acid anhydride; glutamic acid-γ-benzyl ester N-carboxy acid anhydride; ε-trifluoroacetyl group-lysine N-carboxy acid anhydride etc.
Operable amino acid whose residue is abridged according to the code of following 3 letters: alanine (Ala); arginine (Arg); aspartic acid (Asp); asparagine (Asn); halfcystine (Cys); glutamic acid (Glu); glutamine (Gln); glycocoll (Gly); histidine (His); isoleucine (Ile); leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe); serine (Ser); threonine (Thr); tryptophane (Trp); tyrosine (Tyr) and valine (Val), the amino acid with nucleophilic side chain advantageously uses them to be protected on side chain before in the method according to the invention.
Statement " protecting group " is interpreted as referring to the group of any kind, and it stops coupled atom or group (for example oxygen atom or nitrogen-atoms) to participate in undesirable reaction in building-up process.These protecting groups comprise the group of Side chain protective group and protection C-or N-end, are referred to as amine protecting group and sour protecting group.
As the example of the non-limit of amine protecting group, can mention particularly benzoyl (Bz), acetyl group (Ac), trifluoroacetyl group (Tfa), benzyloxycarbonyl group (Z), to benzyloxycarbonylchloride base (2ClZ), to bromo-benzyloxycarbonyl (2BrZ), to nitro benzyloxycarbonyl group, hexichol methoxyl benzyloxy carbonyl, hexichol methoxycarbonyl group, 9-fluorenylmethyloxycarbonyl (Fmoc), tertbutyloxycarbonyl (Boc), benzenesulfonyl, p-toluenesulfonyl or 2-nitrobenzene sulfonyl.
Example as the non-limit of sour protecting group; can mention alkyl, aryl, aralkyl, silicyl type; as methoxy, methylthiomethyl, 2; 2; 2-three chloroethyls, 2-halogenated ethyl, 2-(TMS)-ethyl, the tert-butyl group, aryl, alkyl, aralkyl, allyl, phenyl, trityl (trityl), benzhydryl, to nitrobenzyl, to the group of methoxybenzyl and trialkylsilkl, as trimethyl silicane ethers, triethylsilyl, t-butyldimethylsilyl or isopropyl dimetylsilyl.
For the purposes of the present invention, term " peptide " refers to a kind of polymkeric substance, and the monomer in this polymkeric substance is the amino acid that the covalent bond by amide type links together.Peptide comprises at least two amino acid.Preferably, the amino acid whose number in the peptide chain is more than or equal to 3.Peptide chain often comprises maximum 100 amino acid.Preferably, amino acid whose number is less than or equals 20 in the peptide chain.Particularly preferably, amino acid whose number is less than or equals 15 in the peptide chain.
For the purposes of the present invention, term " polypeptide " refers to that a-amino acid links together with peptide chain and the compound that forms, and it also is the intermediate product of proteolysis.The compound that is formed by two amino acid molecular dehydrating condensations is called dipeptides, in like manner analogizes tripeptides, tetrapeptide, pentapeptide etc. in addition.The common compound that is formed by 10-100 amino acid molecular dehydrating condensation is polypeptide.
For the purposes of the present invention, term " high purity N-carboxy acid anhydride " refers to N-carboxy acid anhydride purity 〉=99.5%, chlorion＜0.01%, free aminoacid content＜0.01%.
For the purposes of the present invention, term " high-purity mixtures of polypeptides " refers to the mixtures of polypeptides that prepared by raw material high purity N-carboxy acid anhydride (purity 〉=99.5%, chlorion＜0.01%, free aminoacid content＜0.01%).
In the present invention, abbreviation " NCA " expression amino acid, n-carboxyanhydrides.
The invention still further relates to following technical proposals:
1. the method for chloride ion content in measuring N-carboxy acid anhydride, it comprises the steps:
(1). preparation can be used for the solution of chloride ion content in measuring N-carboxy acid anhydride, and it comprises:
(a) the N-carboxy acid anhydride is dissolved in one or more substantially anhydrous solvents,
(b) hydrolysis of use hydrolysing agent is dissolved in the N-carboxy acid anhydride in the solvent,
(c) obtain can be used for the solution of chloride ion content in measuring N-carboxy acid anhydride,
(2). measure chloride ion content in the above-mentioned N-carboxy acid anhydride that obtains.
2. according to the method for technical scheme 1, wherein substantially anhydrous solvent is selected from as follows:
-the solvent miscible with water, wherein, solvent is preferably selected from dimethyl sulfoxide (DMSO) (DMSO), N, dinethylformamide (DMF), N, N-dimethyl acetamide (DMA), N-methyl-2 pyrrolidone (NMP), dimethyl sulfoxide (DMSO) (DMSO), formamide and sulfolane, dioxane, tetrahydrofuran (THF), acetone, acetonitrile and carboxylic acid such as formic acid, acetic acid; And/or
-ionic liquid, for example liquid salt of alkylation imidazoles.
3. according to each method of technique scheme, wherein, under inert gas shielding, the N-carboxy acid anhydride is dissolved in one or more substantially anhydrous solvents.
4. according to each method of technique scheme, wherein, use hydrolysing agent such as water or aqueous alkali to be hydrolyzed, preferred water or sodium hydrate aqueous solution, in particular, deionized water.
5. according to each method of technique scheme, wherein, hydrolysis is dissolved in the process of the N-carboxy acid anhydride in the solvent, and the use amount of hydrolysing agent such as water or aqueous alkali is the 0.1-10mL/mgN-carboxy acid anhydride.
6. the solution that can be used for chloride ion content in measuring N-carboxy acid anhydride, it is prepared by the method that comprises the steps:
(a) the N-carboxy acid anhydride is dissolved in one or more substantially anhydrous solvents,
(b) hydrolysis of use hydrolysing agent is dissolved in the N-carboxy acid anhydride in the solvent.
7. according to the method for technical scheme 6, wherein substantially anhydrous solvent is selected from as follows:
-the solvent miscible with water, wherein, solvent is preferably selected from dimethyl sulfoxide (DMSO) (DMSO), N, dinethylformamide (DMF), N, N-dimethyl acetamide (DMA), N-methyl-2 pyrrolidone (NMP), dimethyl sulfoxide (DMSO) (DMSO), formamide and sulfolane, dioxane, tetrahydrofuran (THF), acetone, acetonitrile and carboxylic acid such as formic acid, acetic acid; And/or
-ionic liquid, for example liquid salt of alkylation imidazoles.
8. according to each method of technique scheme 6-7, wherein, under inert gas shielding, the N-carboxy acid anhydride is dissolved in one or more substantially anhydrous solvents.
9. according to each method of technique scheme 6-8, wherein, use hydrolysing agent such as water or aqueous alkali to be hydrolyzed, preferred water or sodium hydrate aqueous solution, in particular, deionized water.
10. according to each method of technique scheme 6-9, wherein, hydrolysis is dissolved in the process of the N-carboxy acid anhydride in the solvent, and the use amount of hydrolysing agent such as water or aqueous alkali is the 0.1-10mL/mgN-carboxy acid anhydride.
11. prepare the method for N-carboxy acid anhydride, it comprises the steps:
A. following selection N-carboxy acid anhydride, wherein chloride ion content is preferably greater than 0.01wt% greater than 0.05wt%:
Chloride ion content in the N-carboxy acid anhydride that-detection obtains;
(1). preparation can be used for the solution of chloride ion content in measuring N-carboxy acid anhydride, and it comprises:
(a) the N-carboxy acid anhydride is dissolved in one or more solvents,
(b) hydrolysis of use hydrolysing agent is dissolved in the N-carboxy acid anhydride in the solvent, obtains can be used for the solution of chloride ion content in measuring N-carboxy acid anhydride;
(2). measure chloride ion content in the above-mentioned N-carboxy acid anhydride that obtains;
When chloride ion content greater than 0.05wt%, when being preferably greater than 0.01wt%, then carrying out purification step and make its chloride ion content less than 0.05wt%, preferably less than 0.01wt%.
12. according to the method for technical scheme 11, wherein substantially anhydrous solvent is selected from as follows:
-the solvent miscible with water, wherein, solvent is preferably selected from dimethyl sulfoxide (DMSO) (DMSO), N, dinethylformamide (DMF), N, N-dimethyl acetamide (DMA), N-methyl-2 pyrrolidone (NMP), dimethyl sulfoxide (DMSO) (DMSO), formamide and sulfolane, dioxane, tetrahydrofuran (THF), acetone, acetonitrile and carboxylic acid such as formic acid, acetic acid; And/or
-ionic liquid, for example liquid salt of alkylation imidazoles.
13. according to each method of technique scheme 11-12, wherein, under inert gas shielding, the N-carboxy acid anhydride is dissolved in one or more substantially anhydrous solvents.
14. according to each method of technique scheme 11-13, wherein, use hydrolysing agent such as water or aqueous alkali to be hydrolyzed, preferred water or sodium hydrate aqueous solution, in particular, deionized water.
15. according to each method of technique scheme 11-14, wherein, hydrolysis is dissolved in the process of the N-carboxy acid anhydride in the solvent, the use amount of described hydrolysing agent such as water or aqueous alkali is: the 0.1-50mL/mgN-carboxy acid anhydride, preferred 1-30mL/mg N-carboxy acid anhydride, especially 1-20mL/mg N-carboxy acid anhydride, more preferably 1-10mL/mg N-carboxy acid anhydride.
16. prepare the method for polypeptide, it comprises the steps:
A. following selection N-carboxy acid anhydride, wherein chloride ion content is preferably greater than 0.01wt% greater than 0.05wt%:
Chloride ion content in the N-carboxy acid anhydride that-detection obtains;
(1). preparation can be used for the solution of chloride ion content in measuring N-carboxy acid anhydride, and it comprises:
(a) the N-carboxy acid anhydride is dissolved in one or more solvents,
(b) hydrolysis of use hydrolysing agent is dissolved in the N-carboxy acid anhydride in the solvent, obtains can be used for the solution of chloride ion content in measuring N-carboxy acid anhydride;
(2). measure chloride ion content in the above-mentioned N-carboxy acid anhydride that obtains;
When chloride ion content greater than 0.05wt%, when being preferably greater than 0.01wt%, then carrying out purification step and make its chloride ion content less than 0.05wt%, preferably less than 0.01wt%.
17. according to the method for technical scheme 16, wherein polypeptide is that the acetic acid lattice draw for thunder.
18. according to each method of technical scheme 16-17, wherein substantially anhydrous solvent is selected from as follows:
-the solvent miscible with water, wherein, solvent is preferably selected from dimethyl sulfoxide (DMSO) (DMSO), N, dinethylformamide (DMF), N, N-dimethyl acetamide (DMA), N-methyl-2 pyrrolidone (NMP), dimethyl sulfoxide (DMSO) (DMSO), formamide and sulfolane, dioxane, tetrahydrofuran (THF), acetone, acetonitrile and carboxylic acid such as formic acid, acetic acid; And/or
-ionic liquid, for example liquid salt of alkylation imidazoles.
19. according to each method of technique scheme 16-18, wherein, under inert gas shielding, the N-carboxy acid anhydride is dissolved in one or more substantially anhydrous solvents.
20. according to each method of technique scheme 16-19, wherein, use hydrolysing agent such as water or aqueous alkali to be hydrolyzed, preferred water or sodium hydrate aqueous solution, in particular, deionized water.
21. according to each method of technique scheme 16-20, wherein, hydrolysis is dissolved in the process of the N-carboxy acid anhydride in the solvent, the use amount of described hydrolysing agent such as water or aqueous alkali is: the 0.1-50mL/mgN-carboxy acid anhydride, preferred 1-30mL/mg N-carboxy acid anhydride, especially 1-20mL/mg N-carboxy acid anhydride, more preferably 1-10mL/mg N-carboxy acid anhydride.
Following example is intended to illustrate the present invention and unrestricted the present invention.
Embodiment
The preparation of embodiment 1. test L-alanine-NCA sample solutions
Accurately take by weighing about 20mg L-alanine-NCA sample to be checked under the nitrogen protection, with 1 milliliter anhydrous 1,4-dioxane (water cut≤0.04%) dissolving.This drips of solution is added in 10 ml deionized water in the stirring, this potpourri is transferred in 20 milliliters of volumetric flasks fully with deionized water, use the deionized water constant volume.
The preparation of embodiment 2. test L-tyrosine-NCA sample solutions
Accurately take by weighing about 20mg L-tyrosine-NCA sample to be checked under the nitrogen protection, with 1 milliliter anhydrous 1,4-dioxane (water cut≤0.04%) dissolving.This drips of solution is added in 10 milliliters of 0.01M sodium hydrate aqueous solutions in the stirring, this potpourri is transferred in 20 milliliters of volumetric flasks fully with deionized water, use the deionized water constant volume.
The preparation of embodiment 3. test L-glutamic acid-γ-benzyl ester-NCA sample solutions
Accurately take by weighing about 20mg L-glutamic acid-γ-benzyl ester-NCA sample to be checked under the nitrogen protection, with 1 milliliter anhydrous 1,4-dioxane (water cut≤0.04%) dissolving.This drips of solution is added in 10 ml deionized water in the stirring, this potpourri is transferred in 20 milliliters of volumetric flasks fully with deionized water, use the deionized water constant volume.
The preparation of embodiment 4. test L-ε-trifluoroacetyl group-lysine-NCA sample solutions
Accurately take by weighing about 20mg L-ε-trifluoroacetyl group-lysine-NCA sample to be checked under the nitrogen protection, with 1 milliliter anhydrous 1,4-dioxane (water cut≤0.04%) dissolving.This drips of solution is added in 10 ml deionized water in the stirring, this potpourri is transferred in 20 milliliters of volumetric flasks fully with deionized water, use the deionized water constant volume.
The preparation of embodiment 5. blank solutions
With 1 milliliter anhydrous 1,4-dioxane (water cut≤0.04%) is added drop-wise in 10 ml deionized water in the stirring, and this potpourri is transferred in 20 milliliters of volumetric flasks fully with deionized water, uses the deionized water constant volume.
Embodiment 6. sample detection
Above-mentioned four solution that prepare are detected with chromatography of ions, and chromatographic column is Dionex IonPac
TMAS19,4.0 * 250mm, flow velocity 1.0ml/min, the KOH of 10mM carries out wash-out, adopts the electric conductivity detector detection signal, and the chlorion appearance time is about the 10.6min place.Prepare reference substance with sodium chloride simultaneously, detect with external standard method.Chlorion testing result such as table 1.
Table 1: chlorion testing result
Embodiment 7. preparation copolymer-1s (that is, the acetic acid lattice draw for thunder)
Add in the there-necked flask through sodium metal handled 1,4-dioxane 200mL adds L-alanine NCA2.590 gram under the room temperature, L-tyrosine NCA1.036 gram, L-glutamic acid-γ-benzyl ester NCA1.974 restrains, L-ε-trifluoroacetyl group-lysine NCA4.693 restrains.Stirred 30 minutes, and to the system clarification, added 33 milligrams of diethylamine.20-25 degree centigrade of following mechanical raking 24 hours.Reactant liquor is slowly poured in the 400mL water, produced a large amount of white solids, behind the suction filtration, vacuum drying gets 7.424 grams, yield 92.8%.
Add above-mentioned shielded lattice in the there-necked flask and draw for thunder 7.0 grams, 31% hydrogen bromide/acetic acid mixed liquor 140mL stirred 22 hours down at 22-24 degree centigrade.The rufous reactant liquor is poured in the 400mL water, produced a large amount of white solids, suction filtration, vacuum drying gets the trifluoroacetyl group lattice and draws for thunder 4.76 grams.Add above-mentioned trifluoroacetyl group lattice in the there-necked flask and draw for thunder 0.76 gram, 1M piperidines aqueous solution 38mL stirred 24 hours under the room temperature.Filter membrane with 1KDa is dialysed, the solution ice acetic acid that obtains to pH be 5.5-5.8, stirred 1 hour.Freeze-drying gets white powder, and mean molecular weight is 7803.
Claims (10)
1. the method for chloride ion content in measuring N-carboxy acid anhydride, it comprises the steps:
(1). preparation can be used for the solution of chloride ion content in measuring N-carboxy acid anhydride, and it comprises:
(a) the N-carboxy acid anhydride is dissolved in one or more substantially anhydrous solvents,
(b) hydrolysis of use hydrolysing agent is dissolved in the N-carboxy acid anhydride in the solvent,
(c) obtain can be used for the solution of chloride ion content in measuring N-carboxy acid anhydride,
(2). measure chloride ion content in the above-mentioned N-carboxy acid anhydride that obtains.
2. as follows according to the process of claim 1 wherein that substantially anhydrous solvent is selected from:
-the solvent miscible with water, wherein, solvent is preferably selected from dimethyl sulfoxide (DMSO) DMSO, N, dinethylformamide DMF, N, N-dimethyl acetamide DMA, N-methyl-2 pyrrolidone NMP, dimethyl sulfoxide (DMSO) DMSO, formamide and sulfolane, dioxane, tetrahydrofuran THF, acetone, acetonitrile and carboxylic acid such as formic acid, acetic acid; And/or
-ionic liquid, for example liquid salt of alkylation imidazoles.
3. require each method according to aforesaid right, wherein, under inert gas shielding, the N-carboxy acid anhydride is dissolved in one or more substantially anhydrous solvents.
4. require each method according to aforesaid right, wherein, use hydrolysing agent such as water or aqueous alkali to be hydrolyzed, preferred water or sodium hydrate aqueous solution, in particular, deionized water.
5. require each method according to aforesaid right, wherein, hydrolysis is dissolved in the process of the N-carboxy acid anhydride in the solvent, and the use amount of described hydrolysing agent such as water or aqueous alkali is 0.1-10mL/mg N-carboxy acid anhydride.
6. the solution that can be used for chloride ion content in measuring N-carboxy acid anhydride, it is prepared by the method that comprises the steps:
(a) the N-carboxy acid anhydride is dissolved in one or more substantially anhydrous solvents,
(b) hydrolysis of use hydrolysing agent is dissolved in the N-carboxy acid anhydride in the solvent.
7. according to the method for claim 6, wherein substantially anhydrous solvent is selected from as follows:
-the solvent miscible with water, wherein, solvent is preferably selected from dimethyl sulfoxide (DMSO) DMSO, N, dinethylformamide DMF, N, N-dimethyl acetamide DMA, N-methyl-2 pyrrolidone NMP, dimethyl sulfoxide (DMSO) DMSO, formamide and sulfolane, dioxane, tetrahydrofuran THF, acetone, acetonitrile and carboxylic acid such as formic acid, acetic acid; And/or-ionic liquid, for example liquid salt of alkylation imidazoles.
8. require each method of 6-7 according to aforesaid right, wherein, under inert gas shielding, the N-carboxy acid anhydride is dissolved in one or more substantially anhydrous solvents.
9. require each method of 6-8 according to aforesaid right, wherein, use hydrolysing agent such as water or aqueous alkali to be hydrolyzed, preferred water or sodium hydrate aqueous solution, in particular, deionized water.
10. require each method of 6-9 according to aforesaid right, wherein, hydrolysis is dissolved in the process of the N-carboxy acid anhydride in the solvent, and the use amount of described hydrolysing agent such as water or aqueous alkali is 0.1-10mL/mg N-carboxy acid anhydride.
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