CN106324153A - Method for measuring content and optical purity of amino acid-N-formic anhydride - Google Patents
Method for measuring content and optical purity of amino acid-N-formic anhydride Download PDFInfo
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Abstract
The invention relates to a method for measuring the content and optical purity of amino acid-N-formic anhydride. The method comprises the following steps: (1) using organic amine to make amino acid NCA carry out derivation to obtain amide with a stable chemical property; and (2) using an external standard method to measure the amide content so as to derive out the content of NCA.
Description
Technical field
The invention belongs to biochemicals detection field, measure aminoacid-N-formic anhydride content and the method for optical purity in particular to a kind of.
Background technology
Acetic acid copaxone be a kind of for treat multiple sclerosis artificial synthetic polypeptide mixture (M.M.Mouradain,
Pharmacology&Therapeutics,98,245-255,2003).Acetic acid copaxone (being again copolymer-1) be one by alanine,
Glutamic acid, lysine and the random. copolymers of tyrosine composition.Its amino acid molar ratio 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.
Acetic acid copaxone is by ALANINE, TYR, Pidolidone-γ-benzyl ester, L-ε-trifluoroacetyl group-lysine, these four amino
The N-formic anhydride (NCA) of acid is polymerized generation at random.And the quality of this polyreaction and polymerizate is for amino acid N CA added
Quality is very sensitive.If we have found that if amino acid N CA can not reach there is specification, the mean molecule quantity of acetic acid copaxone and aminoacid group
Cheng Douhui does not reaches due standard.In order to make acetic acid copaxone can reach standard, the standard of amino acid N CA is content >=95%, optical voidness
Degree >=99.5%.So, the method accurately measuring amino acid N CA content and optical purity is the most particularly important.
Amino acid N CA is 3-substituted isoxazoline-2,5-diketone, and its character is similar to the inner-acid anhydride in organic compound, is embodied in: activity is high,
Meet water unstable, cause difficulty to detection.And four amino acid N CA, i.e. ALANINE NCA, TYR NCA, Pidolidone-γ-benzyl ester
NCA, L-ε-trifluoroacetyl group-lysine NCA, the detection difficulty with ALANINE NCA is maximum again.Because ALANINE NCA activity is the highest, do not have
Having uv absorption, boiling point is low, but impurity boiling point is high.Activity is high, causes and is difficult to get to standard substance, so cannot external standard content；Impurity boiling point is high,
Cause and can not detect purity with gas chromatography；Hydrolabil, causes and can not detect by reversed phase high-performance liquid chromatography；There is no uv absorption, make
Become and select detector aspect cannot use UV-detector；Boiling point is low, causes and cannot use evaporative light detector (ELSD).The method of nuclear-magnetism can detect
The purity of amino acid N CA, but the roughening of its data determines the method is not suitable for purity detecting.
Summary of the invention
Present invention firstly relates to a kind of method measuring aminoacid-N-formic anhydride (amino acid N CA) content, the method comprises the steps,
Amino acid N CA is derived by step (1) with organic amine, obtains the amide of stable chemical nature；
The step (2) content by external standard method amide, derives the content of NCA.
The invention still further relates to a kind of method measuring aminoacid-N-formic anhydride (amino acid N CA) optical purity, the method comprises the steps,
Amino acid N CA is derived by step (1) with chirality organic amine, obtains the amide of stable chemical nature；
The step (2) content by external standard method amide, derives the optical purity of NCA.
Described derivative organic amine is: benzylamine, R-alpha-methylbenzylamine, S-alpha-methylbenzylamine, R-alpha-ethyl benzyl amine, S-alpha-
One or more of ethyl benzyl amine.
Described derivative experiment parameter is, solvent selects: oxolane, 2-methyltetrahydrofuran, Isosorbide-5-Nitrae-dioxane, ethyl acetate, acetic acid are different
Propyl ester, dichloromethane, chloroform；Reaction temperature: room temperature, 15-30 DEG C, the response time: 0.5～2 hour.
Accompanying drawing explanation
Fig. 1, embodiment 1 standard substance 2S-2-Amino-N-benzyl propionic acid amide. external standard curve
Fig. 2, embodiment 2 standard substance (2S-2-amino-N-(R-1-phenylethyl) propionic acid Trifluoroacetate) mass spectrum and nuclear magnetic spectrogram
Fig. 3, embodiment 3 standard substance (2S-2-amino-N-(S-1-phenylethyl) propionic acid Trifluoroacetate) mass spectrum and nuclear magnetic spectrogram
Fig. 4, embodiment 4 standard substance (2S-2-amino-3-(4-hydroxy phenyl)-N-(S-1-phenylethyl) propionic acid Trifluoroacetate) mass spectrum
Figure and nuclear magnetic spectrogram
Fig. 5, embodiment 5 standard substance (4S-4-amino-4-(1-S-1-phenyi-ethylamino carbonyl)-benzyl butyrate trifluoroacetate) mass spectrum
With nuclear magnetic spectrogram
Fig. 6, embodiment 6 standard substance (2S-2-amino-6-(2,2,2-trifluoroacetyl group amino)-N-(S-phenethyl) caproamide trifluoroacetate)
Mass spectrum and nuclear magnetic spectrogram
Fig. 7, embodiment 7 standard substance (2S-2-amino-N-(R-1-phenyl propyl) propionic acid Trifluoroacetate) mass spectrum and nuclear magnetic spectrogram
Fig. 8, embodiment 8 standard substance (2S-2-amino-N-(S-1-phenyl propyl) propionic acid Trifluoroacetate) mass spectrum and nuclear magnetic spectrogram
Detailed description of the invention
The benzylamine derivatives experiment of embodiment 1.L-alanine NCA
Accurate weighing about 20mg ALANINE NCA measuring samples, joins in 1mL oxolane, is added drop-wise to gained solution equipped with 2g benzylamine
With in the there-necked flask of 20 milliliters of oxolanes, it is stirred at room temperature 1 hour.Product is: 2S-2-amino-N '-benzyl propionic acid amide..
The derivative experiment of the R-alpha-methylbenzylamine of embodiment 2.L-alanine NCA
Accurate weighing about 20mg ALANINE NCA measuring samples, joins in 1mL isopropyl acetate, is added drop-wise to gained solution equipped with 2g
In the there-necked flask of R-alpha-methylbenzylamine and 20 milliliters of isopropyl acetates, it is stirred at room temperature 1 hour.Product is: 2S-2-amino-N '-(R-1-
Phenylethyl) propionic acid amide., mass spectrum is shown in Fig. 2.
The derivative experiment of the S-alpha-methylbenzylamine of embodiment 3.L-alanine NCA
Accurate weighing about 20mg ALANINE NCA measuring samples, joins in 1mL dichloromethane, is added drop-wise to gained solution equipped with 2g
In the there-necked flask of S-alpha-methylbenzylamine and 20 milliliters of dichloromethane, it is stirred at room temperature 1 hour.Product is: 2S-2-amino-N '-(S-1-benzene
Base ethyl) propionic acid amide., mass spectrum is shown in Fig. 3.
The derivative experiment of the S-alpha-methylbenzylamine of embodiment 4.L-tyrosine NCA
Accurate weighing about 20mg TYR NCA measuring samples, joins in 1mL2-methyltetrahydrofuran, gained solution is added drop-wise to equipped with
In the there-necked flask of 2g S-alpha-methylbenzylamine and 20 milliliters of 2-methyltetrahydrofurans, it is stirred at room temperature 1 hour.Product is: 2S-2-amino-3-
(4-hydroxy phenyl)-N '-(S-1-phenylethyl) propionic acid amide., mass spectrum is shown in Fig. 4.
The derivative experiment of the S-alpha-methylbenzylamine of embodiment 5.L-glutamic acid-γ-benzyl ester NCA
Accurate weighing about 20mg Pidolidone-γ-benzyl ester NCA measuring samples, joins in 1mL1,4-dioxane, is added drop-wise to by gained solution
In there-necked flask equipped with 2g S-alpha-methylbenzylamine and 20 milliliters of Isosorbide-5-Nitrae-dioxane, it is stirred at room temperature 1 hour.Product is: 4S-4-amino-4-
(1-S-1-phenyi-ethylamino carbonyl)-benzyl butyrate, mass spectrum is shown in Fig. 5.
The derivative experiment of the S-alpha-methylbenzylamine of embodiment 6.L-ε-trifluoroacetyl group-lysine NCA
Accurate weighing about 20mg L-ε-trifluoroacetyl group-lysine NCA measuring samples, joins in 1mL ethyl acetate, is dripped by gained solution
In the there-necked flask equipped with 2g S-alpha-methylbenzylamine and 20 milliliters of ethyl acetate, it is stirred at room temperature 1 hour.Product is: 2S-2-amino-6-
(2,2,2-trifluoroacetyl group amino)-N '-(S-1-phenethyl) caproamide, mass spectrum is shown in Fig. 6.
The derivative experiment of the R-alpha-ethyl benzyl amine of embodiment 7.L-alanine NCA
Accurate weighing about 20mg ALANINE NCA measuring samples, joins in 1mL dichloromethane, is added drop-wise to gained solution equipped with 2g
In the there-necked flask of R-alpha-ethyl benzyl amine and 20 milliliters of dichloromethane, it is stirred at room temperature 1 hour.Product is: 2S-2-amino-N '-(R-1-benzene
Base propyl group) propionic acid amide., mass spectrum is shown in Fig. 7.
The derivative experiment of the S-alpha-ethyl benzyl amine of embodiment 8.L-alanine NCA
Accurate weighing about 20mg ALANINE NCA measuring samples, joins in 1mL chloroform, is added drop-wise to gained solution equipped with 2g S-alpha-
In the there-necked flask of ethyl benzyl amine and 20 milliliters of chloroforms, it is stirred at room temperature 1 hour.Product is: 2S-2-amino-N '-(S-1-phenyl propyl) propionyl
Amine, mass spectrum is shown in Fig. 8.
The preparation of embodiment 9. standard substance
Embodiment 1 standard substance: 2S-2-Amino-N-benzyl propionic acid amide., No. CAS: 1214706-98-5, buying is from Matrix Scientific.
Standard substance are typically prepared method
There-necked flask adds 30 grams of Fmoc-L-Ala-OH, dichloromethane 500 milliliters, benzylamine or alpha substituted benzylamine 15 grams, HATU30 gram, diisopropyl
Base ethylamine 20 milliliters, stirs 2 hours under room temperature.After some plate confirms that reaction terminates, add purified water 200 milliliters.Separate organic facies, anhydrous
Sodium sulfate is spin-dried for after drying.Cross chromatographic column to purify.
The amide obtained is added in oxolane and dissolves, add 5 equivalent hexahydropyridines, stir 24 hours under room temperature.After completion of the reaction, rotate
It is evaporated to dryness, prepares standard substance by liquid chromatograph.
Embodiment 2 standard substance: 2S-2-amino-N-(R-1-phenylethyl) propionic acid Trifluoroacetate, ESI-MS m/z (M+1)=193.1；1H NMR (400MHz, DMSO) δ 8.83 (d, J=7.9Hz, 1H), 8.11 (s, 3H), 7.36 7.30 (m, 4H), 7.27-
7.12 (m, 1H), 4.92 (p, J=7.0Hz, 1H), 3.82 (s, 3H), 1.38 (t, J=6.5Hz, 6H).
Embodiment 3 standard substance: 2S-2-amino-N-(S-1-phenylethyl) propionic acid Trifluoroacetate, ESI-MS m/z (M+1)=193.0；1H NMR (400MHz, DMSO) δ 8.84 (d, J=7.8Hz, 1H), 8.12 (s, 3H), 7.41 7.27 (m, 4H), 7.27
7.19 (m, 1H), 4.94 (p, J=7.1Hz, 1H), 3.87 (s, 1H), 1.39 (d, J=7.0Hz, 3H), 1.33 (d, J=
7.0Hz,3H).
Embodiment 4 standard substance: 2S-2-amino-3-(4-hydroxy phenyl)-N-(S-1-phenylethyl) propionic acid Trifluoroacetate, ESI-MS m/z
(M+1)=285.2；1H NMR (400MHz, DMSO) δ 9.37 (s, 1H), 8.75 (d, J=7.8Hz, 1H), 8.15 (s, 3H),
7.35 7.17 (m, 3H), 7.04 (d, J=7.1Hz, 2H), 6.92 (d, J=8.3Hz, 2H), 6.64 (d, J=8.3Hz,
2H), 4.91 (p, J=6.9Hz, 1H), 3.92 (t, J=6.9Hz, 1H), 2.86 (ddd, J=29.5,14.0,7.4Hz, 2H),
1.37 (d, J=7.0Hz, 3H).
Embodiment 5 standard substance: 4S-4-amino-4-(1-S-1-phenyi-ethylamino carbonyl)-benzyl butyrate trifluoroacetate, ESI-MS m/z (M+1)
=341.2；1H NMR (400MHz, DMSO) δ 8.93 (d, J=7.7Hz, 1H), 8.16 (s, 3H), 7.45 7.16 (m, 10H),
5.08 (s, 2H), 4.94 (p, J=6.9Hz, 1H), 3.83 (s, 1H), 2.41 2.16 (m, 2H), 2.08 1.88 (m,
2H), 1.39 (d, J=7.0Hz, 3H).
Embodiment 6 standard substance: 2S-2-amino-6-(2,2,2-trifluoroacetyl group amino)-N-(S-phenethyl) caproamide trifluoroacetate, ESI-MS
M/z (M+1)=346.3；1H NMR (400MHz, DMSO) δ 9.39 (s, 1H), 8.87 (d, J=7.7Hz, 1H), 8.09 (s, 3H),
7.50 7.03 (m, 5H), 4.95 (p, J=6.9Hz, 1H), 3.75 (dd, J=13.0,8.3Hz, 1H), 3.09 (dd, J
=13.3,6.6Hz, 2H), 1.68 (dd, J=14.6,7.4Hz, 2H), 1.54 1.34 (m, 4H), 1.32 1.05 (m,
3H).
Embodiment 7 standard substance: 2S-2-amino-N-(R-1-phenyl propyl) propionic acid Trifluoroacetate, ESI-MS m/z (M+1)=207.2；1H NMR (400MHz, DMSO) δ 8.76 (d, J=8.5Hz, 1H), 8.08 (s, 3H), 7.38 7.16 (m, 5H), 4.72 (dd,
J=15.0,8.3Hz, 1H), 3.85 (s, 1H), 1.80 1.61 (m, 2H), 1.41 (d, J=7.0Hz, 3H), 0.85 (t,
J=7.3Hz, 3H).
Embodiment 8 standard substance: 2S-2-amino-N-(S-1-phenyl propyl) propionic acid Trifluoroacetate, ESI-MS m/z (M+1)=207.0；1H NMR (400MHz, DMSO) δ 8.74 (d, J=8.0Hz, 1H), 8.07 (s, 3H), 7.38 7.18 (m, 5H), 4.70 (q,
J=7.5Hz, 1H), 3.90 (s, 1H), 1.71 (p, J=7.3Hz, 2H), 1.31 (d, J=7.0Hz, 3H), 0.86 (t, J
=7.3Hz, 3H).
Embodiment 10. sample detection
Outer marking quantitative
Solution is prepared:
Take reference substance 2S-2-amino-N-(R-1-phenylethyl) propionic acid amide. 58.96mg, add isopropyl acetate and dissolve, and be settled to 20ml, make
For reference substance storing solution A.
50% contrast solution: precision pipettes comparison storing solution 1.25ml, puts in 5ml measuring bottle, adds isopropyl acetate and be diluted to scale, shake up, to obtain final product.
80% contrast solution: precision pipettes comparison storing solution 2.0ml, puts in 5ml measuring bottle, adds isopropyl acetate and be diluted to scale, shake up, to obtain final product.
100% contrast solution: precision pipettes comparison storing solution 2.5ml, puts in 5ml measuring bottle, adds isopropyl acetate and be diluted to scale, shake up, to obtain final product.
120% contrast solution: precision pipettes comparison storing solution 3.0ml, puts in 5ml measuring bottle, adds isopropyl acetate and be diluted to scale, shake up, to obtain final product.
150% contrast solution: precision pipettes comparison storing solution 3.75ml, puts in 5ml measuring bottle, adds isopropyl acetate and be diluted to scale, shake up, to obtain final product.
Sample solution is prepared: by embodiment 2 method, take ALANINE NCA 20mg, accurately weighed, joins in 1mL isopropyl acetate, will
Gained solution is added drop-wise in the there-necked flask equipped with 2g R-alpha-methylbenzylamine and 20 milliliters of isopropyl acetates, is stirred at room temperature 1 hour, to obtain final product.
Take above-mentioned contrast solution and embodiment 2 sample solution by following chromatographic condition sample introduction analysis
Chromatographic column Kromasil 100-5C18250 × 4.6mm
Detector: UV, 280nm
Flowing phase: acetonitrile: water=30:70
Flow velocity: 1.0ml/min
Column temperature: 30 DEG C
Sample size: 10ul
Control test result and external standard calibration trace are prepared such as following table, standard curve such as Fig. 1
Sample solution testing result
| ALANINE NCA mg | Sample solution peak area | Concentration mg/ml | Content |
| 20.12 | 1048585 | 1.5916 | 99.5% |
External standard positions
Solution is prepared
Take reference substance 2R-2-amino-N-(R-1-phenylethyl) propionic acid amide. 58.88mg, add isopropyl acetate and dissolve, and be settled to 20ml, make
For reference substance storing solution B.
Contrast solution: take comparison storing solution A 2.5ml, compare storing solution B 0.25ml, be placed in 5ml volumetric flask, add isopropyl acetate and be diluted to
Scale, mix homogeneously, to obtain final product.
Take above-mentioned contrast solution and embodiment 2 sample solution by following chromatographic condition sample introduction analysis
Chromatographic column Kromasil 100-5C18250 × 4.6mm
Detector: UV, 280nm
Flowing phase: acetonitrile: water=30:70
Flow velocity: 1.0ml/min
Column temperature: 30 DEG C
Sample size: 10ul
Utilize the difference of different optics chirality reference substances in contrast solution to go out peak position, L-type in sample solution and D type product are carried out chromatograph location.
Thus extrapolating the optical purity of ALANINE NCA in embodiment 2 is 99.8%.The reactant liquor that will obtain in embodiment 1-8, by above-mentioned external standard
Quantitatively and external standard localization method, sample detection is directly carried out.
Amino acid N CA content and optical purity testing result in embodiment 1-8:
| Amino acid N CA | Lot number | Content detection result | Optical purity result |
| ALANINE NCA | Embodiment 1 | 96.1% | Cannot detect |
| ALANINE NCA | Embodiment 2 | 99.5% | 99.8% |
| ALANINE NCA | Embodiment 3 | 101.2% | 100.0% |
| TYR NCA | Embodiment 4 | 100.3% | 99.8% |
| Pidolidone-γ-benzyl ester NCA | Embodiment 5 | 95.8% | 100.0% |
| L-ε-trifluoroacetyl group-lysine NCA | Embodiment 6 | 95.4% | 100.0% |
| ALANINE NCA | Embodiment 7 | 95.9% | 100.0% |
| ALANINE NCA | Embodiment 8 | 95.4% | 99.8% |
Last it should be noted that above example is only used for helping skilled in the art to understand the essence of the present invention, the present invention being not used as protects
Protect the concrete restriction of scope.
Claims (9)
1. the method measuring aminoacid-N-formic anhydride (amino acid N CA) content, the method comprises the steps,
Amino acid N CA is derived by step (1) with organic amine, obtains the amide compound of stable chemical nature；
The step (2) content by external standard method amide, derives the content of NCA.
2. the method measuring aminoacid-N-formic anhydride (amino acid N CA) optical purity, the method comprises the steps,
Amino acid N CA is derived by step (1) with chirality organic amine, obtains the amide compound of stable chemical nature；
The step (2) content by external standard method amide, derives the optical purity of NCA.
3. according to the arbitrary described method of claim 1 or 2, it is characterised in that derivative organic amine is: benzylamine, R-alpha-methylbenzylamine, S-alpha-
One or more in methylbenzylamine, R-alpha-ethyl benzyl amine or S-alpha-ethyl benzyl amine.
4. according to the arbitrary described method of claim 1-3, it is characterised in that the method that amino acid N CA is derived by described step 1) organic amine
Become testing sample solution for being dissolved with organic solvent by testing sample, testing sample solution is added drop-wise in the there-necked flask equipped with organic amine solution, stirs
Mix reaction, it is thus achieved that stable amide compound solution is directly used in step 2) detection；
Preferably, described organic solution is selected from: oxolane, 2-methyltetrahydrofuran, Isosorbide-5-Nitrae-dioxane, ethyl acetate, isopropyl acetate, dichloro
One or more in methane, chloroform；Described organic amine solution be organic amine be dissolved in oxolane, 2-methyltetrahydrofuran, 1,4-dioxane,
In one or more solvents in ethyl acetate, isopropyl acetate, dichloromethane, chloroform.
Reaction temperature: room temperature or 15-30 DEG C；
Response time: 0.5～2 hour.
5. according to the arbitrary described method of claim 1-4, it is characterised in that described aminoacid-N-formic anhydride is ALANINE NCA, TYR NCA,
One or more in Pidolidone-γ-benzyl ester NCA, L-ε-trifluoroacetyl group-lysine NCA.
6. according to the arbitrary described method of claim 1-5, it is characterised in that described stable amide compound is the amino of organic amine and amino acid whose alphe
The amide that carboxyl reaction obtains.
7. according to the arbitrary described method of claim 1-6, it is characterised in that step 2) it is that the method with HPLC is to step 1) gained amide compound
Thing solution carries out detection by quantitative.
Method the most according to claim 7, it is characterised in that chromatographic condition is chromatographic column Kromasil 100-5C18 250 × 4.6mm, detection
Device: UV, 280nm, flow phase: acetonitrile: water=30:70, flow velocity: 1.0ml/min, column temperature: 30 DEG C, sample size: 10ul.
9. according to the arbitrary described method of claim 1-8, it is characterised in that step 2) passed through by HPLC external standard method acquisition amide compound quality
Molal weight obtains the amino acid N CA molal quantity of testing sample, and then obtains content and optical purity.
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| CN103335873A (en) * | 2013-06-04 | 2013-10-02 | 深圳翰宇药业股份有限公司 | Method for measuring chloride ion content of amino acid-N-formic anhydride |
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| CN103335873A (en) * | 2013-06-04 | 2013-10-02 | 深圳翰宇药业股份有限公司 | Method for measuring chloride ion content of amino acid-N-formic anhydride |
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| Title |
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| TAMAS VAJDA ET AL.: "CRYO-BIOORGANIC CHEMISTRY: FREEZING EFFECT ON STEREOSELECTION OF DL-ALANINE-N-CARBOXYANHYDRIDE OLIGOMERIZATION IN DIOXANE SOLUTION", 《CRYOLETTERS》 * |
| 丁永胜 等: "氨基酸的分析方法及其应用进展", 《色谱》 * |
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