CN103159823A - Folic-acid-modified peptide synthesis technique - Google Patents

Abstract

The invention discloses a folic-acid-modified peptide synthesis technique, belonging to the field of medicine production. The synthesis technique comprises the following steps: 1) swelling with resin; 2) connecting a first amino acid; 3) deprotecting; 4) detecting; 5) washing; 6) condensing; 7) washing; 8) repeating the operations from the step 2) to the step 6) to sequentially connect amino acids in the sequence from right to left; 9) weighing 5 times of equivalent weight of folic acid, adding into a reaction tube; adding 5 times of equivalent weight of HBTU and 10 times of equivalent weight of DIEA (diisopropylethylamine), reacting for 30 minutes, removing the reaction solution, taking the resin, and carrying out ninhydrin detection; 10) cutting polypeptide from the resin for 120 minutes; 11) blow-drying, and washing; 12) purifying the polypeptide by HPLC (high performance liquid chromatography); 13) freeze-drying the purified solution to obtain the finished product; 14) identification: carrying out molecular weight identification of MS and purity identification by HPLC analysis; and 15) carrying out sealed packaging on the white powdered polypeptide, and storing at -20 DEG C. The invention enhances the success rate in the traditional technique; and since the connection of the polypeptide and folic acid is synchronously completed in the synthesis process, the labor cost is greatly lowered, and the efficiency is greatly enhanced.

Description

Modified with folic acid peptide synthesis technique

Technical field

The present invention relates to a kind of modified with folic acid peptide synthesis technique of medical production field.

Background technology

One of folic acid (Folic acid) vitamin B complex is equivalent to VitB11 (pteroylglutamic acid, PGA), is that Mitchell (H.K.Mitchell, 1941) extracts purifying from the leaf of spinach, so called after folic acid.The effect that promotes juvenile cell maturation in marrow is arranged, can cause megaloblastic anemia and leukopenia if the mankind lack folic acid, the pregnant woman is even more important.Peptide is that a-amino acid links together with peptide chain and the compound that forms, and it is also 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.Usually the compound that is formed by 10 ~ 100 amino acid molecular dehydrating condensations is polypeptide.Their molecular weight is lower than 10,000Da(Dalton dalton), can see through semi-permeable membranes, do not precipitated by trichoroacetic acid(TCA) and ammonium sulfate.Also there is document that the peptide that is comprised of 2 ~ 10 amino acid is called oligopeptides (small-molecular peptides); The peptide of 10 ~ 50 amino acid compositions is called polypeptide; The peptide that is comprised of the amino acid more than 50 just is called protein.At present the research about the functional aspect of the combination of folic acid and polypeptide is a focus direction.But the method to the coupling of folic acid and polypeptide on market still is in the junior stage, and technique of the present invention is to have made optimization on forefathers' Research foundation.Can effectively improve the success ratio of coupling and reach yield, reducing synthetic cost.

Summary of the invention

The objective of the invention is to overcome the deficiencies in the prior art, optimize existing technical process, a kind of success ratio that can effectively improve coupling, and yield and reduce the modified with folic acid peptide new synthetic process of synthetic cost are provided.

The present invention is achieved by the following technical solutions:

A kind of modified with folic acid peptide synthesis technique, its synthesis technique step is as follows:

1) resin swelling

2-Chlorotrityl Chloride Resin resin is put into reaction tubes, add DCM (15ml/g), vibration 30min;

2) connect first amino acid

Fall solvent by husky core suction filtration, add the Fmoc-L-Pro-OH amino acid of 3 times of molar excess, then add the DIEA of 10 times of molar excess, add at last the DMF dissolving, vibration 30min;

3) deprotection

Remove DMF, add 20% piperidines DMF solution (15ml/g), 5min removes and adds 20% piperidines DMF solution (15ml/g), 15min again;

4) detect

Take out piperidine solution, get tens grainy resins, wash three times with ethanol, add triketohydrindene hydrate, KCN, each of phenol solution, 105 ℃-110 ℃ heating 5min deepen blue positive reaction;

5) wash

DMF (10ml/g) twice, methyl alcohol (10ml/g) twice, twice of DMF (10ml/g);

6) condensation

Method a: three times of protected amino acids (FmocAAA-OH) are excessive, and three times of HBTU are excessive, all with DMF dissolving less as far as possible, add reaction tubes, add at once ten times of NMM excessive. reaction 30min;

7) wash DMF (10ml/g) once, methyl alcohol (10ml/g) twice, twice of DMF (10ml/g); 8) repeat the operation of two to seven steps, from right to left the amino acid in catenation sequence successively;

9) 5 times of equivalent folic acid of weighing, join in reaction tubes, then adds the HBTU of 5 times of equivalents, the DIEA of 10 times of equivalents, and reaction 30min had both taken out reaction solution, got resin and carried out the triketohydrindene hydrate detection;

10) cut polypeptide from resin

Preparation cutting liquid (10/g) TFA 94.5%; Water 2.5%; EDT 2.5%; TIS 1%;

Clipping time: 120min;

11) dry up washing

Lysate is dried up with nitrogen as far as possible, wash six times with ether, then normal temperature volatilizes;

12) use the HPLC purified polypeptide;

13) at last with the solution freeze-drying after purifying, both obtained finished product;

14) identify

The finished product polypeptide that takes a morsel is respectively done the molecular weight identification of MS, and the Purity analyzed of HPLC;

15) with the polypeptide of white powder, pack-20 ℃ of preservations.

The present invention compared with prior art has following beneficial effect:

1. improve the success ratio of coupling and reach yield;

2. reduce synthetic cost.

Embodiment

Below in conjunction with embodiment, content of the present invention is described in further detail:

technique of the present invention is on the basis of the solid phase synthesis of polypeptide, utilize the synthetic advantage of Solid-phase Polypeptide, practical situation in conjunction with folic acid, and a kind of modified with folic acid peptide synthesis technique that proposes, at first be the polypeptide portion sequence of definite folic acid peptide that will synthesize, then the method according to solid phase synthesis is synthesized polypeptide, but first not cracking, only out get final product the end NH2 of polypeptide is exposed, then according to the actual molar equivalent of polypeptide, ratio with folic acid: polypeptide=5:1 drops into reaction with folic acid, then can detect very easily with triketohydrindene hydrate--whether NH2 exists can judge whether reaction is complete.This technique has improved the upper success ratio of traditional technology greatly, and also owing to being that polypeptide is connected connection and is synchronously completed in synthetic with folic acid, old friend's work cost has also reduced greatly, and efficient is greatly improved.

Be below synthesis material and related reagent:

1) protected amino acid raw material

Fmoc-AAA-OH, folic acid

2) condensation reagent

HBTU，DIEA

3) solvent

DMF, DCM, acetonitrile

4) resin

2-Chlorotrityl?Chloride?Resin

5) deprotecting regent

Piperidines

6) cutting reagent

TFA，TIS，EDT?，H2O

7) nitrogen

8) anhydrous diethyl ether

9) precise electronic balance

The synthesis technique step is as follows:

1) resin swelling

2-Chlorotrityl Chloride Resin resin is put into reaction tubes, add DCM (15ml/g), vibration 30min;

2) connect first amino acid

Fall solvent by husky core suction filtration, add the Fmoc-L-Pro-OH amino acid of 3 times of molar excess, then add the DIEA of 10 times of molar excess, add at last the DMF dissolving, vibration 30min;

3) deprotection

Remove DMF, add 20% piperidines DMF solution (15ml/g), 5min removes and adds 20% piperidines DMF solution (15ml/g), 15min again;

4) detect

Take out piperidine solution, get tens grainy resins, wash three times with ethanol, add triketohydrindene hydrate, KCN, each of phenol solution, 105 ℃-110 ℃ heating 5min deepen blue positive reaction;

5) wash

DMF (10ml/g) twice, methyl alcohol (10ml/g) twice, twice of DMF (10ml/g)

6) condensation

Method a: three times of protected amino acids (FmocAAA-OH) are excessive, and three times of HBTU are excessive, all with DMF dissolving less as far as possible, add reaction tubes, add at once ten times of NMM excessive. reaction 30min;

7) wash

DMF (10ml/g) once, methyl alcohol (10ml/g) twice, twice of DMF (10ml/g); 8) repeat the operation of two to seven steps, from right to left the amino acid in catenation sequence successively;

9) 5 times of equivalent folic acid of weighing, join in reaction tubes, then adds the HBTU of 5 times of equivalents, the DIEA of 10 times of equivalents, and reaction 30min had both taken out reaction solution, got resin and carried out the triketohydrindene hydrate detection;

10) cut polypeptide from resin

Preparation cutting liquid (10/g) TFA 94.5%; Water 2.5%; EDT 2.5%; TIS 1%;

Clipping time: 120min;

11) dry up washing

Lysate is dried up with nitrogen as far as possible, wash six times with ether, then normal temperature volatilizes;

12) use the HPLC purified polypeptide;

13) at last with the solution freeze-drying after purifying, both obtained finished product;

14) identify

The finished product polypeptide that takes a morsel is respectively done the molecular weight identification of MS, and the Purity analyzed of HPLC;

15) with the polypeptide of white powder, pack-20 ℃ of preservations.

Claims (1)

1. modified with folic acid peptide synthesis technique, it is characterized in that: the synthesis technique step is as follows:

1) resin swelling

2-Chlorotrityl Chloride Resin resin is put into reaction tubes, add DCM (15ml/g), vibration 30min;

2) connect first amino acid

Fall solvent by husky core suction filtration, add the Fmoc-L-Pro-OH amino acid of 3 times of molar excess, then add the DIEA of 10 times of molar excess, add at last the DMF dissolving, vibration 30min;

3) deprotection

Remove DMF, add 20% piperidines DMF solution (15ml/g), 5min removes and adds 20% piperidines DMF solution (15ml/g), 15min again;

4) detect

Take out piperidine solution, get tens grainy resins, wash three times with ethanol, add triketohydrindene hydrate, KCN, each of phenol solution, 105 ℃-110 ℃ heating 5min deepen blue positive reaction;

5) wash

DMF (10ml/g) twice, methyl alcohol (10ml/g) twice, twice of DMF (10ml/g);

6) condensation

Method a: three times of protected amino acids (FmocAAA-OH) are excessive, and three times of HBTU are excessive, all with DMF dissolving less as far as possible, add reaction tubes, add at once ten times of NMM excessive. reaction 30min;

7) wash

DMF (10ml/g) once, methyl alcohol (10ml/g) twice, twice of DMF (10ml/g); 8) repeat the operation of two to seven steps, from right to left the amino acid in catenation sequence successively;

9) 5 times of equivalent folic acid of weighing, join in reaction tubes, then adds the HBTU of 5 times of equivalents, the DIEA of 10 times of equivalents, and reaction 30min had both taken out reaction solution, got resin and carried out the triketohydrindene hydrate detection;

10) cut polypeptide from resin

Preparation cutting liquid (10/g) TFA 94.5%, water 2.5%, EDT 2.5%, and TIS 1%;

Clipping time: 120min;

11) dry up washing

Lysate is dried up with nitrogen as far as possible, wash six times with ether, then normal temperature volatilizes;

12) use the HPLC purified polypeptide;

13) at last with the solution freeze-drying after purifying, both obtained finished product;

14) identify

The finished product polypeptide that takes a morsel is respectively done the molecular weight identification of MS, and the Purity analyzed of HPLC;

15) with the polypeptide of white powder, pack-20 ℃ of preservations.