CN106916086A - A kind of solid phase synthesis process of paranitroanilinum modified polypeptide C-terminal - Google Patents
A kind of solid phase synthesis process of paranitroanilinum modified polypeptide C-terminal Download PDFInfo
- Publication number
- CN106916086A CN106916086A CN201510999419.7A CN201510999419A CN106916086A CN 106916086 A CN106916086 A CN 106916086A CN 201510999419 A CN201510999419 A CN 201510999419A CN 106916086 A CN106916086 A CN 106916086A
- Authority
- CN
- China
- Prior art keywords
- paranitroanilinum
- amino acid
- pna
- fmoc
- resin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 0 CC(C)(C)*C(CC(*)C(Nc(cc1)ccc1[N+]([O-])=O)=O)=O Chemical compound CC(C)(C)*C(CC(*)C(Nc(cc1)ccc1[N+]([O-])=O)=O)=O 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C269/00—Preparation of derivatives of carbamic acid, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
- C07C269/06—Preparation of derivatives of carbamic acid, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups by reactions not involving the formation of carbamate groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
- C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The present invention relates to a kind of solid phase synthesis process of paranitroanilinum modified polypeptide C-terminal, it comprises the following steps：A) amino acid of paranitroanilinum modified alpha carboxyl is prepared；B) amino acid side chain and resin are coupled；C) solid phase synthesis process coupling amino acid one by one, forms peptide resin；D) cracking of peptide resin；E) RPLC purifying.Wherein step a) is that the amino acid of protection and paranitroanilinum are condensed in the presence of condensing agent; obtain crude product; gained crude product is washed with acid and/or alkaline aqueous solution and neutral aqueous solution, is entered without carrying out other purification steps and is sloughed side chain protected step.
Description
Technical field
The present invention relates to pharmaceutical synthesis field, and in particular to the synthetic method of the amine-modified polypeptide of p-nitrophenyl.
Background technology
It is the polypeptide quasi-molecule that a class has pharmaceutical activity containing the amine-modified polypeptide of p-nitrophenyl, is various ammonia peptides
One important synthesis substrate of enzyme family member activity test.Aminopeptidase (Aminopeptidases) is used as a kind of
Proteolytic enzyme, can be from free N-terminal (amino terminal) ecto-entad of polypeptide chain hydrolysis amino acid one by one.These
Aminopeptidase not only regulation human normal physiological function in terms of play an important role, and turned into medical diagnosis on disease with
The target for the treatment of, has extensive use in field of biomedical research.In drug research field, paranitroanilinum
The polypeptide of modification to being played an important role in the propagation of malignant cell, differentiation, invasion and attack and angiogenesis,
It is an important target for studying antineoplastic.
With the development of medical technology and going deep into for research, development synthesis C-terminal is by the amine-modified polypeptide of p-nitrophenyl
Method tool is of great significance.Current rarer document and patent report synthesis paranitroanilinum modified polypeptide C
The method at end.
Nitro has very strong electron-withdrawing power in p-nitrophenyl amine molecule, and nitro and amino are in the right of phenyl ring
Position, it is very slow in the process for forming amido link, cause reaction yield to reduce.Few pertinent literature reports are closed
In the synthesis of L- asparagus fern ammonia pNA, Pidolidone pNA etc..In experimentation find peptide molecule directly with to nitre
The yield of base aniline reaction is general all than relatively low.During peptide modified, with the increase of peptide molecule, repair
The degree of difficulty of decorations has great importance also with increasing, the method that development efficiently synthesizes pNA modified polypeptides.
Document (chemical reagent, 2012,34 (3), 269-271) report synthesis pNA amino acid is frequently with phosgene
Method synthesizes, and phosgene is obtained corresponding NCA intermediates by the method with amino acid effect first, then by NCA
Intermediate obtains the amino acid of pNA modifications with pNA reactions；Because the toxicity of phosgene is big, the technique passes through improvement,
Carrying out reaction using the less triphosgene of toxicity can also obtain corresponding pNA modified amino acids.Although technique pair
Yield increases, but triphosgene or phosgene all have larger toxicity, and to equipment, environment,
Operating personnel have violent injury.Had no for the document that peptide molecule directly carries out pNA modifications with triphosgene
Report.PNA modification yields are carried out using conventional full guard polypeptide fragment and is very lowly not easy to purifying, finally
Cost is caused to steeply rise, the further research to medical science brings difficulty.
The content of the invention
In order to overcome above mentioned problem, the present invention use solid-phase synthesis, are with cheap Fmoc protected amino acids
Raw material, first from liquid phase reactor prepare corresponding Fmoc-Glu (OH)-pNA,
Fmoc-Asp (OH)-pNA, Fmoc-Ser (OH)-pNA, Fmoc-Thr (OH)-pNA, the side through recrystallizing
Method obtains pNA amino acid starting material of the purity more than 95%, then carries out amino acid using the method for synthesis in solid state
Condensation, most obtains the thick peptide of target through cracking afterwards.The thick peptide purity obtained using the method is higher, is easy to liquid-phase pure
Change, the method greatly reduces production cost.
The present invention is comprised the following steps：
1) preparation of pNA amino acid monomers
2) coupling of amino acid side chain and resin；
3) solid phase synthesis process is coupled one by one；
4) cracking of peptide resin；
5) RPLC purifying；
The step 1) pNA modifications amino acid protect using Fmoc of amino acid through condensation reaction, go guarantor
Two step synthesis of shield reaction；
The full guard pNA amino acid that the step a) step of condensation is obtained need not use recrystallization purifying,
Protective reaction is directly carried out by only needing to simple pickling, alkali cleaning；
In step a) the protective reaction steps, the crude product for obtaining is recrystallized can obtain purity 95%
The exposed pNA modified amino acids of side chain above；
The method for connecing resin using amino acid side chain in the step b) is coupled；
Connecing resin for Fmoc-Glu (OH)-pNA, Fmoc-Asp (OH)-pNA in the step b) can be with
Selection Wang resins and 2-CTC chlorine resins；
In the step b) Choice of Resin is connect for Fmoc-Ser (OH)-pNA, Fmoc-Thr (OH)-pNA
2-CTC chlorine resins；
Fmoc-Ser (OH)-pNA, Fmoc-Thr (OH)-pNA connection Choice of Resin in the step b)
2-CTC chlorine resins use DiPEA+CsCO3Method, the conditioned response of nitrogen atmosphere 24 hours；
The step 2) in Fmoc-Glu (OH)-pNA, Fmoc-Asp (COOH)-pNA connect resin using normal
The coupling method of rule；It is HOBt or HOAt that connection Wang resins use DIC+A or B+A+C, wherein A,
B is HBTU, HATU, TBTU, PyAOP or PyBOP, and C is DIPEA or TMP；Connection 2-CTC
Chlorine resin uses DiPEA methods；
The step 3) in coupling using DIC+A or B+A+C method be condensed, reaction to ninhydrin detection
Resin transparent；Wherein A be HOBt or HOAt, B be HBTU, HATU, TBTU, PyAOP or
PyBOP, C are DIPEA or TMP；
The step 4) the middle TFA for cracking use different ratio：PhSMe：EDT：PhOMe：H2O is molten
Liquid.
Concrete technology flow process is as follows：
This hair prepares the amine-modified Fmoc amino acid of p-nitrophenyl using the method for liquid phase synthesis first, by weight
The method of crystallization can be very good control and then the side chain of amino acid be connected on resin, and order is coupled, finally
Cracking, purifying obtain the amine-modified peptide molecule long of p-nitrophenyl.It is amine-modified using this method solve p-nitrophenyl
The technical problem of peptide long, the amine-modified peptide long of Fmoc Solid phase synthesis p-nitrophenyls have low cost, be easy to it is pure
Change, the small peptide product purity of wherein pNA modifications can reach more than 99%.
Synthesis route：By taking Fmoc-Asp (OtBu)-OH as an example：
One aspect of the present invention provides a kind of preparation method of the amino acid of paranitroanilinum modified alpha carboxyl,
1) the α amino and pendant reactive group of amino acid are protected,
2) by step 1) gained protection amino acid be condensed in the presence of condensing agent with paranitroanilinum,
Crude product is obtained, gained crude product is washed with acid and/or alkaline aqueous solution and neutral aqueous solution, without
Carry out other purification steps and enter step 3),
3) by step 2) obtained by crude product slough side chain protected,
4) by step 3) obtained by slough side chain protected crude product dissolving, regulation pH value for acidity, go forward side by side
Row recrystallization purifying.
Further, the pendant reactive group is hydroxyl or carboxyl, and side chain protected is selected from-tBu.
Further, step 2) in method of condensing select DIC+A or B+A+C, wherein A be HOBt
Or HOAt, B are HBTU, HATU, TBTU, PyAOP or PyBOP, C is DIPEA or TMP
Method.Wherein DIC+A combination mol ratios 1-1.5:1-1.5, B+A+C combine mol ratio
1-1.5:1-1.5:1.5-2、；Preferably DIC+HOAt, DIPEA+HOAt+HATU system, the protection
The mol ratio of amino acid and paranitroanilinum be 1:1-1.5(1:1.1), it is highly preferred that being first catalyzed with condensing agent
Amino acid, then adds paranitroanilinum；It is aqueous hydrochloric acid solution, alkali that acid solution is used in last handling process
Property solution be that saturated sodium bicarbonate or sodium carbonate liquor, neutral solution are the saturated common salt aqueous solution.
Further, wherein step 3) it is with TFA/CH2Cl2Solution slough the reaction of side chain protected, instead
Organic solvent is removed after should stopping, surplus solution is poured into frozen water be filtrated to get and is sloughed the thick of side chain protected
Product；Preferably, reaction temperature is 20-30 DEG C (room temperature), and the reaction time is 1-3 hours (2 hours).
Further, wherein step 4) in pH value adjust to 5-6, and with ethyl acetate, methyl tertbutyl
Ether, acetone, dichloromethane, methyl alcohol, ethanol equal solvent or its mixed solvent are recrystallized.
Another aspect of the present invention provides a kind of solid phase synthesis process of paranitroanilinum modified polypeptide C-terminal,
It comprises the following steps：
A) method according to claim any one of 1-5 prepares the amino acid of paranitroanilinum modified alpha carboxyl；
B) by the coupling of amino acid side chain and resin；
C) solid phase synthesis process coupling amino acid one by one, forms peptide resin；
D) cracking of peptide resin；
E) RPLC purifying.
Further, the solid phase synthesis process of paranitroanilinum modified polypeptide C-terminal according to claim 6,
Wherein,
In step b) Choice of Resin Wang is met for Fmoc-Glu (OH)-pNA, Fmoc-Asp (OH)-pNA
Resin or 2-CTC chlorine resins；Preferably, connection Wang resins use DIC+A or B+A+C, wherein
A is HOBt or HOAt, B are HBTU, HATU, TBTU, PyAOP or PyBOP, and C is DIPEA
Or TMP；Connection 2-CTC chlorine resin uses DiPEA methods；
Choice of Resin 2-CTC chlorine resins are connect for Fmoc-Ser (OH)-pNA, Fmoc-Thr (OH)-pNA；
It is preferred that using DiPEA+CsCO3Method, the conditioned response of nitrogen atmosphere 24 hours；
Further, step 3) in coupling be condensed using the method for DIC+A or B+A+C, reaction is to indenes three
Ketone detects resin transparent；Wherein A is HOBt or HOAt, B are HBTU, HATU, TBTU, PyAOP
Or PyBOP, C are DIPEA or TMP.
Further, step 4) the middle TFA for cracking use different ratio：PhSMe：EDT：PhOMe：
H2O solution.
Brief description of the drawings
Fig. 1 is the mass spectrogram of the products therefrom of embodiment 2.
Fig. 2 is the mass spectrogram of the products therefrom of embodiment 5.
Fig. 3 is the mass spectrogram of the products therefrom of embodiment 8.
Fig. 4 is the mass spectrogram of the products therefrom of embodiment 11.
Specific embodiment
Embodiment is given below to be specifically described with to the present invention, it is necessary to it is pointed out here that be following examples
It is served only for that the present invention is further illustrated, it is impossible to be interpreted as limiting the scope of the invention, the field
Person skilled in the art according to some nonessential modifications and adaptations that present invention is made to the present invention, still belong to
In protection scope of the present invention.
Embodiment 1：Side chain protected, the synthesis of the Asp that main chain is modified by pNA
400mL dichloromethane, Fmoc-Asp (OtBu) 42.2g (102mmol) are added in 1L reaction bulbs,
Stirring makes raw material all dissolve；Solution clarify add in backward reaction bulb HOAt 16.7g (122mmol) and
DIC 19ml (123mmol) continue to stir 30min, then to addition paranitroanilinum (pNA) in solution
15.5g (115mmol) room temperature reaction 24 hours；Added water in the backward reaction bulb of reaction stopping and reaction is quenched, according to
It is secondary washed with 2mol/L hydrochloric acid solutions twice, saturated sodium carbonate solution wash twice and saturated common salt water washing one time；
Organic phase anhydrous sodium sulfate drying, is concentrated under reduced pressure to give Fmoc-Asp (OtBu)-pNA crude products.
Embodiment 2：Side chain is exposed, the synthesis of the Asp that main chain is modified by pNA
Fmoc-Asp (the OtBu)-pNA crude product 400mL concentration obtained in embodiment 1 is 80%
TFA/CH2Cl2(volume ratio) solution carries out sloughing tBu reactions.Room temperature reaction two hours, reaction is depressurized after stopping
Most of dichloromethane solution is removed, surplus solution is poured into 500mL frozen water, be filtrated to get
Fmoc-Asp (OH)-pNA crude products；Crude product is dissolved with dichloromethane, saturated sodium bicarbonate washing crude product solution is straight
Produced to without obvious bubble, water phase pH is adjusted to 5-6 with 1mol/L hydrochloric acid, separate organic phase using anhydrous sulphur
Sour sodium is dried, and be concentrated under reduced pressure to obtain Fmoc-Asp (OH)-pNA, and ethyl acetate carries out recrystallizing to obtain product to crude product
30.1g, reaction yield 62%.Product result product after mass spectrum and nuclear-magnetism confirm recrystallization is
Fmoc-Asp(OH)-pNA；MS：[M+H]=476.17:；1H-NMR(DMSO-d6, 400M) and ppm, δ
7.69 (d, J=3.9Hz, 2H), 7.56 (d, J=8.8Hz, 4H), 7.40 (d, J=8.8Hz, 4H), 7.18 (d,
J=4.0Hz, 2H), 4.84 (d, J=5.6Hz, 2H), 3.85 (d, J=6.3Hz, 2H), 3.70 (t, J=5.6
Hz, 1H), 3.61 (t, J=6.3Hz, 1H)
13C NMR(100MHz,DMSO-d6)δ165.94,160.79,154.07,138.43,137.05,
136.47(2C),133.25(2C),128.42(2C),128.10(4C),125.90(4C),118.32(2C),
71.30,67.71,63.45,48.99.
Embodiment 3：The reaction of Fmoc-Asp (OH)-pNA and resin
It is the 2-CTC chlorine resins of 0.485mmol/g to weigh 41.2g substitution degrees, appropriate DMF washings 3 times,
Swelling 30min is stand-by；Add embodiment 2 in obtain 28.5g (60mmol) Fmoc-Asp (OH)-pNA,
DIPEA 15.5g (120mmol), appropriate DMF react 3 hours, reaction added after terminating methyl alcohol 15ml,
DIPEA 7.8g (60mmol) continue to react 30 minutes, and reaction terminates rear DMF and washs 3 times, that is, obtain
Fmoc-Asp(-2-CTC Resin)-pNA。
Embodiment 4：The synthesis of peptide resin
Fmoc-Asp (- 2-CTC the Resin)-pNA resins that will be obtained in embodiment 3 continue on for synthesizing pNA
The polypeptide of modification.Can be coupled in following manner in the coupling of subsequent amino-acid, until resin is used
Ninhydrin detection is transparent.The activator of subsequent amino-acid is DIC+A or B+A+C, and wherein A is HOBt
Or HOAt, B are HBTU, HATU, TBTU, PyAOP or PyBOP, C is DIPEA or TMP.
In the method once coupling Fmoc-Val-OH, Fmoc-Glu (OtBu)-OH and
Fmoc-Asp (OtBu)-OH, Fmoc is removed after the completion of coupling using 20% Piperidine/DMF solution, and DMF is washed
Wash more than 6 times, methyl alcohol shrinks, vacuum drying obtains peptide resin 53g, waits to crack.
Embodiment 5：The cracking of peptide resin
The lytic reagent that the cracking of pNA modification peptide resins can be used is TFA：PhSMe：PhOMe：EDT：
H2O：TIS=80~90:0~5:0~3:0~5:0~5：0~2 (V:V), TFA is selected in experiment：PhSMe：
PhOMe：EDT：H2O=85:5:3:5:2.It is existing to being added in the 53g peptide resins for obtaining in embodiment 4
The lysate 530ml for matching somebody with somebody, is stirred at room temperature reaction 2.5 hours, and reaction is filtered to remove resin after terminating, filtrate uses
The sedimentation of 5L frost ether, centrifugation, drying under reduced pressure obtain the thick peptide 12.1g of pNA, and thick peptide purity is produced close to 90%
Thing result passes through mass spectrum MS：[M-H]=595.44.
Embodiment 6：HPLC purifying containing the thick peptides of pNA
The pNA for obtaining is aoxidized in Example 5 and modifies thick peptide, filtration under diminished pressure removes insoluble matter, using Waters
2545RP-HPLC systems, wavelength 210nm, chromatographic column is the anti-phase C18 posts of 50 × 250mm, column temperature
It it is 38 DEG C, conventional 0.1%TFA/ water/acetonitrile mobile phase purifying collects purpose peak component, concentrated under reduced pressure, freezing
Purity is dried to obtain more than 98.5% fine peptide 9.8g.
Embodiment 7：Side chain protected, the Asp synthesis that main chain is modified by pNA
380mL dichloromethane, Fmoc-Asp (OtBu) 37.3g (90mmol) are added in 1L reaction bulbs,
Stirring makes raw material all dissolve；Solution adds HOAt 14.7g (108mmol), HATu in clarifying backward reaction bulb
40.3g (106mmol), keeps solution temperature at 0-5 DEG C, then slowly to being slowly added into DIE in solution
29.2ml (180mmol), to addition paranitroanilinum (pNA) 13.7g (100mmol) in solution after 30min
Room temperature reaction 24 hours；Added water in the backward reaction bulb of reaction stopping and reaction is quenched, it is molten with 2mol/L hydrochloric acid successively
Liquid wash twice, saturated sodium carbonate solution wash twice and saturated common salt water washing one time；The anhydrous sulphur of organic phase
Sour sodium is dried, and is concentrated under reduced pressure to give Fmoc-Asp (OtBu)-pNA crude products.
Embodiment 8：Side chain is exposed, the Asp synthesis that main chain is modified by pNA
Fmoc-Asp (the OtBu)-pNA crude product 400mL concentration obtained in embodiment 7 is 80%
TFA/CH2Cl2(volume ratio V/V) solution carries out sloughing tBu reactions.Room temperature reaction two hours, after reaction stops
Most of dichloromethane solution is removed under reduced pressure, surplus solution is poured into 500mL frozen water, be filtrated to get
Fmoc-Asp (OH)-pNA crude products；Crude product is dissolved with dichloromethane, saturated sodium bicarbonate washing crude product solution is straight
Produced to without obvious bubble, water phase pH is adjusted to 5-6 with 1mol/L hydrochloric acid, separate organic phase using anhydrous sulphur
Sour sodium is dried, and be concentrated under reduced pressure to obtain Fmoc-Asp (OH)-pNA, and ethyl acetate carries out recrystallizing to obtain product to crude product
27.5g, reaction yield 64%.Product result product after mass spectrum and nuclear-magnetism confirm recrystallization is
Fmoc-Asp(OH)-pNA；MS：[M-H]=474.42:；1H-NMR(DMSO-d6, 400M) and ppm,
δ 7.72 (d, J=3.9Hz, 2H), 7.61 (d, J=8.8Hz, 4H), 7.43 (d, J=8.8Hz, 4H),
7.22 (d, J=4.0Hz, 2H), 4.89 (d, J=8.1,5.6Hz, 2H), 3.89 (d, J=6.3Hz, 2H),
3.75 (t, J=6.0Hz, 1H), 3.65 (t, J=5.3Hz, 1H)
13C NMR(100MHz,DMSO-d6)δ166.04,160.89,154.27,138.49,137.16,
136.66(2C),133.32(2C),128.49(2C),128.18(4C),125.99(4C),118.39(2C),
71.39,67.81,63.55,48.97.
Embodiment 9：The reaction of Fmoc-Asp (OH)-pNA and resin
Weigh the Wang fat that 35.2g substitution degrees are 0.512mmol/g, appropriate DMF wash 3 times, it is swelling
30min is stand-by；Fmoc-Asp (OH)-pNA 25.7g (54mmol), HOBt are dissolved with appropriate DMF
7.3g (54mmol), ice bath cooling reaction system keeps solution temperature at 0-5 DEG C, is then slowly added into DIC
8.5g (65mmol), continuation reaction 3 hours in reaction column are poured into after activating 3min, and reaction is used after stopping and fitted
Amount DMF is washed 3 times, adds appropriate acetic anhydride and pyridine mixed solution (volume ratio 7:6) continue to react 3
Hour, reaction terminates rear DMF and washs 6 times, that is, obtain Fmoc-Asp (- Wang Resin)-pNA.
Embodiment 10：The synthesis of peptide resin
Fmoc-Asp (- Wang the Resin)-pNA resins that will be obtained in embodiment 9 continue on for synthesizing pNA
The polypeptide of modification.Can be coupled in following manner in the coupling of subsequent amino-acid, until resin is used
Ninhydrin detection is transparent.The activator of subsequent amino-acid is DIC+A or B+A+C, and wherein A is HOBt
Or HOAt, B are HBTU, HATU, TBTU, PyAOP or PyBOP, C is DIPEA or TMP.
Fmoc-Thr (tBu)-OH, Fmoc-Glu (OtBu)-OH and Fmoc-Ile-OH are once coupled in the method,
Fmoc is removed using 20% Piperidine/DMF solution after the completion of coupling, DMF is washed more than 6 times, and methyl alcohol shrinks,
Vacuum drying obtains peptide resin 48g, waits to crack.
Embodiment 11：The cracking of peptide resin
The lytic reagent that the cracking of pNA modification peptide resins can be used is TFA：PhSMe：PhOMe：EDT：
H2O：TIS=80~90:0~5:0~3:0~5:0~5:0~2 (V:V), TFA is selected in experiment：PhSMe：
PhOMe：EDT：H2O=85:5:3:5:2.It is existing to being added in the 53g peptide resins for obtaining in embodiment 4
The lysate 530ml for matching somebody with somebody, is stirred at room temperature reaction 2.5 hours, and reaction is filtered to remove resin after terminating, filtrate uses
The sedimentation of 5L frost ether, centrifugation, drying under reduced pressure obtain the thick peptide 9.5g of pNA, and thick peptide purity is close to 90%；Produce
Thing result passes through mass spectrum MS：[M-H]=595.54.
Embodiment 12：HPLC purifying containing the thick peptides of pNA
The pNA for obtaining is aoxidized in Example 5 and modifies thick peptide, filtration under diminished pressure removes insoluble matter, using Waters
2545RP-HPLC systems, wavelength 210nm, chromatographic column is the anti-phase C18 posts of 50 × 250mm, column temperature
It it is 38 DEG C, conventional 0.1%TFA/ water/acetonitrile mobile phase purifying collects purpose peak component, concentrated under reduced pressure, freezing
Purity is dried to obtain more than 98.5% fine peptide 7.4g.
Embodiment 13：The synthesis of Boc-Asp (OtBu)-Glu (OtBu)-Val-Asp (OtBu)-OH
It is the 2-CTC chlorine resins of 0.525mmol/g to weigh 57.5g substitution degrees, appropriate DMF washings 3 times,
Swelling 30min is stand-by；Add 37.1g (90mmol) Fmoc-Asp (OtBu)-OH, DIPEA 23.5g
(180mmol), appropriate DMF reacts 3 hours, and reaction adds methyl alcohol 20ml, DIPEA12.4g after terminating
Continue to react 30 minutes, reaction terminates rear DMF and washs 3 times, that is, obtain Fmoc-Asp (OtBu) -2-CTC
Resin。
The activator of subsequent amino-acid is DIC+A or B+A+C, and wherein A is HOBt or HOAt, B are
HBTU, HATU, TBTU, PyAOP or PyBOP, C are DIPEA or TMP.In the method
Fmoc-Val-OH, Fmoc-Glu (OtBu)-OH and Boc-Asp (OtBu)-OH is once coupled, coupling is completed
DMF is washed 3 times afterwards, and methyl alcohol shrinks, and vacuum drying obtains peptide resin 81g, using 1%TFA/CH2Cl2
(V/V) cracking resin obtains full guard peptide 23.7g.
Embodiment 14：The coupling of Boc-Asp (OtBu)-Glu (OtBu)-Val-Asp (OtBu)-OH and pNA
The full guard peptide 230ml dichloromethane dissolving that will be obtained in embodiment 13, adds HOAt 4.12g
After (30mmol) is stirred at room temperature dissolving, DIC 3.88g (30mmol) are slowly added into, after stirring 30min
Add pNA 4.26g (30mmol) room temperature reaction 24 hours.Reaction terminates rear removal of solvent under reduced pressure, fits
Amount ethyl acetate dissolving, successively with 1mol/L HCl solutions, saturation NaHCO3Solution and sodium chloride solution washing,
Anhydrous sodium sulfate drying, inspection is concentrated to give thick peptide 24.9g.Mass spectrum can be found that target peak, and HPLC detections are thick
Full guard peptide and pNA are mainly in peptide.
Thick peptide is obtained into Asp-Glu-Val-Asp-pNA fine peptide 0.585g using HPLC purifying, yield is about
3.3%.
The implication of the abbreviation used in specification and claims is listed in the following table：
Claims (10)
1. a kind of preparation method of the amino acid of paranitroanilinum modified alpha carboxyl,
1) the α amino and pendant reactive group of amino acid are protected,
2) by step 1) gained protection amino acid be condensed in the presence of condensing agent with paranitroanilinum,
Crude product is obtained, gained crude product is washed with acid and/or alkaline aqueous solution and neutral aqueous solution, without
Carry out other purification steps and enter step 3),
3) by step 2) obtained by crude product slough side chain protected,
4) by step 3) obtained by slough side chain protected crude product dissolving, regulation pH value for acidity, go forward side by side
Row recrystallization purifying.
2. the preparation method of the amino acid of paranitroanilinum modified alpha carboxyl according to claim 1, wherein,
The pendant reactive group is hydroxyl or carboxyl, and side chain protected is selected from-tBu, and amino protecting group is selected from Fmoc.
3. the preparation side of the amino acid of the paranitroanilinum modified alpha carboxyl according to claim any one of 1-2
Method, wherein, step 2) in method of condensing select DIC+A or B+A+C, wherein A be HOBt or
HOAt, B are HBTU, HATU, TBTU, PyAOP or PyBOP, and C is DIPEA's or TMP
Method.Wherein DIC+A combination mol ratios 1-1.5:1-1.5, B+A+C combine mol ratio
1-1.5:1-1.5:1.5-2、；Preferably DIC+HOAt, DIPEA+HOAt+HATU system, the protection
The mol ratio of amino acid and paranitroanilinum be 1:1-1.5(1:1.1), it is highly preferred that being first catalyzed with condensing agent
Amino acid, then adds paranitroanilinum；It is aqueous hydrochloric acid solution, alkali that acid solution is used in last handling process
Property solution be that saturated sodium bicarbonate or sodium carbonate liquor, neutral solution are the saturated common salt aqueous solution.
4. the preparation side of the amino acid of the paranitroanilinum modified alpha carboxyl according to claim any one of 1-3
Method, wherein step 3) it is with TFA/CH2Cl2Solution slough the reaction of side chain protected, and reaction is gone after stopping
Except organic solvent, surplus solution is poured into frozen water to carry out being filtrated to get the crude product for sloughing side chain protected；It is preferred that
Ground, reaction temperature is 20-30 DEG C (room temperature), and the reaction time is 1-3 hours (2 hours).
5. the preparation side of the amino acid of the paranitroanilinum modified alpha carboxyl according to claim any one of 1-4
Method, wherein step 4) in pH value adjust to 5-6, and with ethyl acetate, methyl tertiary butyl ether(MTBE), acetone,
Dichloromethane, methyl alcohol, ethanol, tetrahydrofuran equal solvent or its mixed solvent are recrystallized.
6. a kind of solid phase synthesis process of paranitroanilinum modified polypeptide C-terminal, it comprises the following steps：
A) method according to claim any one of 1-5 prepares the amino acid of paranitroanilinum modified alpha carboxyl；
B) by the coupling of amino acid side chain and resin；
C) solid phase synthesis process coupling amino acid one by one, forms peptide resin；
D) cracking of peptide resin；
E) RPLC purifying.
7. the solid phase synthesis process of paranitroanilinum modified polypeptide C-terminal according to claim 6, wherein,
In step b) Choice of Resin Wang is met for Fmoc-Glu (OH)-pNA, Fmoc-Asp (OH)-pNA
Resin or 2-CTC chlorine resins；Preferably, connection Wang resins use DIC+A or B+A+C, wherein
A is HOBt or HOAt, B are HBTU, HATU, TBTU, PyAOP or PyBOP, and C is DIPEA
Or TMP；Connection 2-CTC chlorine resin uses DiPEA methods；
Choice of Resin 2-CTC chlorine resins are connect for Fmoc-Ser (OH)-pNA, Fmoc-Thr (OH)-pNA；
It is preferred that using DiPEA+CsCO3Method, the conditioned response of nitrogen atmosphere 24 hours.
8. the solid phase synthesis process of the paranitroanilinum modified polypeptide C-terminal according to claim any one of 6-7,
Wherein, step 3) in coupling using DIC+A or B+A+C method be condensed, reaction to ninhydrin detect set
Fat is transparent；Wherein A is that HOBt or HOAt, B are HBTU, HATU, TBTU, PyAOP or PyBOP,
C is DIPEA or TMP.
9. the solid phase synthesis process of the paranitroanilinum modified polypeptide C-terminal according to claim any one of 6-8,
Wherein, step 4) the middle TFA for cracking use different ratio：PhSMe：EDT：PhOMe：H2O solution.
10. the synthetic method of the polypeptide of the amine-modified C-terminal of a kind of p-nitrophenyl, it comprises the following steps：
(1) the exposed full guard polypeptide of synthesis C-terminal,
(2) full guard peptide organic solvent (preferably dichloromethane) dissolves, and adds HOAt that dissolving is stirred at room temperature
Afterwards, DIC is slowly added into, pNA is added after stirring 15-60min, room temperature reaction to reaction terminates；
Preferably, HOAt：DIC：The mol ratio of pNA is 1:0.8-1.5:0.8-1.5 (more preferably 1:1:1).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510999419.7A CN106916086A (en) | 2015-12-28 | 2015-12-28 | A kind of solid phase synthesis process of paranitroanilinum modified polypeptide C-terminal |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510999419.7A CN106916086A (en) | 2015-12-28 | 2015-12-28 | A kind of solid phase synthesis process of paranitroanilinum modified polypeptide C-terminal |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106916086A true CN106916086A (en) | 2017-07-04 |
Family
ID=59455846
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510999419.7A Pending CN106916086A (en) | 2015-12-28 | 2015-12-28 | A kind of solid phase synthesis process of paranitroanilinum modified polypeptide C-terminal |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106916086A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111233714A (en) * | 2020-03-18 | 2020-06-05 | 滨海吉尔多肽有限公司 | Preparation method of MAPS polypeptide |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5498695A (en) * | 1992-11-24 | 1996-03-12 | Pfizer Inc. | Paranitroaniline peptides |
| US20050277690A1 (en) * | 2004-06-09 | 2005-12-15 | Sircar Jagadish C | Small molecules for treatment of hypercholesterolemia and related diseases |
| CN102924568A (en) * | 2012-11-20 | 2013-02-13 | 天津铭恒科技发展有限公司 | Synthetic peptides, as well as preparation method and application thereof |
| CN103641889A (en) * | 2013-12-12 | 2014-03-19 | 中国药科大学 | Hypoglycemic peptide and drug use thereof |
| CN104418948A (en) * | 2013-09-10 | 2015-03-18 | 深圳翰宇药业股份有限公司 | Method for preparing polypeptide medicament |
-
2015
- 2015-12-28 CN CN201510999419.7A patent/CN106916086A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5498695A (en) * | 1992-11-24 | 1996-03-12 | Pfizer Inc. | Paranitroaniline peptides |
| US20050277690A1 (en) * | 2004-06-09 | 2005-12-15 | Sircar Jagadish C | Small molecules for treatment of hypercholesterolemia and related diseases |
| CN102924568A (en) * | 2012-11-20 | 2013-02-13 | 天津铭恒科技发展有限公司 | Synthetic peptides, as well as preparation method and application thereof |
| CN104418948A (en) * | 2013-09-10 | 2015-03-18 | 深圳翰宇药业股份有限公司 | Method for preparing polypeptide medicament |
| CN103641889A (en) * | 2013-12-12 | 2014-03-19 | 中国药科大学 | Hypoglycemic peptide and drug use thereof |
Non-Patent Citations (5)
| Title |
|---|
| ANNE BERNHARDT等: ""Solid-phase synthesis of peptide-4-nitroanilides"", 《INT. J. PEPTIDE PROTEIN RES.》 * |
| ANNE BERNHARDT等: ""The solid-phase synthesis of side-chain-phosphorylated peptide-4-nitroanilides"", 《J. PEPTIDE. RES.》 * |
| LAWRENCE A. REITER等: ""Peptidic p-nitroanilide substrates of interleukin-1β-converting enzyme"", 《INT. J. PEPTIDE PROTEIN RES.》 * |
| MICHAEL L. SCHULTE等: ""2-Substituted Nγ-glutamylanilides as novel probes of ASCT2 with improved potency"", 《BIOORGANIC & MEDICINAL CHEMISTRY LETTERS》 * |
| SUDESHNA KAR等: ""Nano structures through self-assembly of protected hydrophobic amino acids: encapsulation of rhodamine B dye by proline-based nanovesicles"", 《J. MATER. SCI.》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111233714A (en) * | 2020-03-18 | 2020-06-05 | 滨海吉尔多肽有限公司 | Preparation method of MAPS polypeptide |
| CN111233714B (en) * | 2020-03-18 | 2022-04-08 | 滨海吉尔多肽有限公司 | Preparation method of MAPS polypeptide |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2421887B1 (en) | Method for the manufacture of degarelix | |
| EP3398957B1 (en) | Method for synthesizing etelcalcetide | |
| CN110590911B (en) | Synthetic method and application of polypeptide containing tyrosine sulfating modification | |
| US10577394B2 (en) | Ganirelix precursor and method for preparing ganirelix acetate by using the same | |
| WO2017097194A1 (en) | Completely-solid-phase preparation method for carbetocin | |
| CN103012563A (en) | Solid-phase synthesis method of antibacterial peptide Iseganan | |
| CN105001298B (en) | A kind of synthesis isolation and purification method of indissoluble polypeptide | |
| CN109734794A (en) | A kind of preparation method of A Bapa peptide | |
| CN106554391B (en) | Method for synthesizing marine biological peptide Xen2174 | |
| CN111499693B (en) | Method for preparing linaclotide by solid-liquid combination | |
| CN105001307B (en) | A kind of coupling peptide chain dissolving indissoluble polypeptide and application isolated and purified in liquid chromatograph thereof | |
| CN113412272A (en) | Improved process for preparing procatide | |
| CN106916086A (en) | A kind of solid phase synthesis process of paranitroanilinum modified polypeptide C-terminal | |
| CN103897029B (en) | A kind of preparation method of romidepsin | |
| CN1865282B (en) | Solid phase polypeptide synthesis preparation method for terlipressin | |
| CN101508724B (en) | Method of preparing tetradecapeptide somatostatin | |
| CN104418948B (en) | A method of preparing polypeptide drugs | |
| WO2021103458A1 (en) | Solid-phase synthesis method for degarelix | |
| CN106317161B (en) | Preparation method of fluoromethyl ketone peptide series compounds | |
| CN108484735A (en) | The synthetic method of a kind of activity ring seven peptide Reniochalistatin A-D extensively | |
| CN110330560B (en) | Synthetic method of lanreotide acetate | |
| CN112062828B (en) | FGF-5 polypeptide inhibitor and hair growth promoting application thereof | |
| WO2019001459A1 (en) | Peptide compound, application thereof and composition containing same | |
| CN108864275B (en) | A kind of synthetic method of Ularitide | |
| Reszka et al. | Control of aspartate epimerization during the coupling of caspase specific tetrapeptides with aromatic amines by using N-[[(dimethylamino)-1H-1, 2, 3-triazolo [4, 5-b]-pyridin-1-yl] methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) as a coupling reagent |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170704 |
|
| RJ01 | Rejection of invention patent application after publication |