CN108129546A - For tryptophan and its highly selective chemical modification method of residue - Google Patents
For tryptophan and its highly selective chemical modification method of residue Download PDFInfo
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- CN108129546A CN108129546A CN201810028218.6A CN201810028218A CN108129546A CN 108129546 A CN108129546 A CN 108129546A CN 201810028218 A CN201810028218 A CN 201810028218A CN 108129546 A CN108129546 A CN 108129546A
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
- C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
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Abstract
The invention discloses a kind of highly selective chemical modification methods for being directed to tryptophan and its residue：Using tryptophan or the substance for containing trp residue as being modified object, Yu Shuizhong, highly selective three components electrophilic substitution reaction occurs for aryl primary amine dressing agent and the tryptophan/trp residue being modified in object under the action of aldehyde component, so as to which aryl primary amine modification agent molecule is connected firmly to covalent bond on tryptophan/trp residue.In the chemical modification method, aryl primary amine dressing agent has excellent selectivity to the tryptophan in protein and its residue, to other active aminos acid and its residue without notable reactivity；Primary amine is modified between agent molecule and tryptophan and its residue and forms covalent bonding, excellent in te pins of durability.
Description
Technical field
The present invention relates to a kind of highly selective chemical modification methods for being directed to tryptophan and its residue.
Background technology
Chemical modification technology is an important directions of modern biotechnology development.It is every by chemical group
It introduces or removes, protein covalent structure is made to change, the chemical modification of protein can be known as.Chemical modification is research
Protein structure and a kind of powerful of a kind of emic important means and directional transformation protein properties.Albumen
There are mainly two types of the chemical modification methods of matter：(1) change the side-chain radical of protein molecule；(2) change the master of protein molecule
Chain structure.Wherein, the reaction modified for the side-chain structure of Amino Acids in Proteins residue is that easy, efficient chemistry is repaiied
Decorations means are capable of the property of engineered protein molecule using the means, such as make its activation, inactivation or assign functional, raising
Stability etc..
Protein is the material base of life, is the main undertaker of vital movement.It is intricate that protein molecule has
Three-dimensional structure, and the side-chain radical of its all amino acid residues is distributed in the various pieces of protein molecule.Some residues
Surface in protein molecule is susceptible to the attack and effect of reagent, the hydrophobic of protein molecule is embedded in some residues
In interior environment, it is not easy to contact and react with reagent.In addition, in different research purposes, to the portion of chemical modification reaction generation
Also there is certain requirement in position.Therefore, for the highly selective chemical modification method of certain amino acid residue in protein structure into
For current research hotspot.
Protein is typically what is be composed of more than 20 kinds of amino acid by different proportion, in numerous amino acid residues,
Tryptophan, tyrosine and histidine residues are since there are aromatic ring or heterocycle structures, and cloud density is higher, easily by electrophilic examination
Electrophilic substitution reaction occurs for the attack of agent, realizes chemical modification.For example, tryptophan, tyrosine and histidine residues can be with
Diazonium reactant salt illustrates that the strong electrophilicity reagent such as diazol has active amino acid good reactivity, but selectivity is not good enough,
It is difficult to realize the chemical modification to particular amino acid residue site.
Invention CN201310737395.9 discloses a kind of covalent bond color method suitable for silk：Group containing aryl primary amine
Dyestuff under the action of aldehyde material in silk tyrosine residue occur Mannich react；The group containing aryl primary amine
Dyestuff and silk mass ratio be 0.005~0.03：1, the dyestuff of the group containing aryl primary amine and aldehyde material mole are
1：1~2；Dye bath ratio is 1：20~100；Value in Dyeing Process is maintained at 4.0~6.5, and dyeing temperature is 30~70 DEG C, soaking time
0.5~24 hour；The patent aryl primary amine dyestuff reaction site is the tyrosine residue in silk.
Invention content
The technical problem to be solved in the present invention is to provide a kind of highly selective chemical modifications for tryptophan and its residue
Method.
In order to solve the above technical problem, the present invention provides a kind of highly selective chemistry for tryptophan and its residue to repair
Decorations method：
Using tryptophan or the substance for containing trp residue as object is modified, Yu Shuizhong is fragrant under the action of aldehyde component
Primary amine dressing agent (dressing agent of the group containing aryl primary amine, abbreviation aryl primary amine dressing agent) and the tryptophan/tryptophan being modified in object
Highly selective three components electrophilic substitution reaction occurs for residue, so as to which aryl primary amine modification agent molecule is connected firmly to color with covalent bond
On propylhomoserin/trp residue.
The improvement for tryptophan and its highly selective chemical modification method of residue as the present invention：The aryl primary amine
The molar ratio of dressing agent and tryptophan/trp residue is 0.1~10：1, the molar ratio of aryl primary amine dressing agent and aldehyde component is 1：
3~100；It was 4.0~6.5 to adjust the pH value of entire reaction system, in 20~60 DEG C of insulation reactions 1~24 hour；Reaction gained
It is dry after product is washed.
Being further improved for the highly selective chemical modification method of tryptophan and its residue as the present invention：Water with
The mass ratio of aryl primary amine dressing agent is 20~7000：1.
Being further improved for the highly selective chemical modification method of tryptophan and its residue as the present invention：It is described
At least need that there are an aryl primary amine segments in the structure of aryl primary amine dressing agent.
It is described as being further improved for the highly selective chemical modification method of tryptophan and its residue of the present invention
Aryl primary amine dressing agent for it is following any one：Aniline, naphthylamines, anil, naphthylamine derivative；
The anil is open-chain crown ether, acetparaminosalol aniline, P-nethoxyaniline, aryl primary amine dyestuff C.I.
Acid brown 4, C.I. acid greens 20.
Being further improved for the highly selective chemical modification method of tryptophan and its residue as the present invention：It is described
Aldehyde component is formaldehyde, acetaldehyde, propionic aldehyde, butyraldehyde, pyroracemic aldehyde, glutaraldehyde or benzaldehyde.
Being further improved for the highly selective chemical modification method of tryptophan and its residue as the present invention：It is repaiied
Jewelry is tryptophan, the polypeptide A containing trp residue, the protein containing trp residue (for example, contain trp residue
Mulberry silk).
In the present invention, above-mentioned pH value is adjusted with the buffer solution of Acetic acid-sodium acetate.
After reaction, pH value is adjusted to 5.0, and product is precipitated, after filtering, washs, dries and (dried in the air at room temperature to constant weight).
The present invention is modified aryl primary amine using three component reactions of aryl primary amine dressing agent, aldehyde component and tryptophan and its residue
Agent, by C-N abutment covalent bondings, is a kind of highly selective chemistry for being directed to tryptophan and its residue with tryptophan and its residue
Method of modifying.Its reaction equation may be as follows：
Heretofore described aryl primary amine dressing agent reaction site is tryptophan and its residue, and tyrosine and its residue experiment are demonstrate,proved
It is real not react on this condition.
Carrying out modification to protein with chemical modification method provided by the present invention has following technical advantage：(1) change
Learn aryl primary amine dressing agent in method of modifying has excellent selectivity to the tryptophan in protein and its residue, active to other
Amino acid and its residue are without notable reactivity；(2) covalent bond is formed between aryl primary amine modification agent molecule and tryptophan and its residue
It closes, excellent in te pins of durability；(3) protein can be chemically modified under room temperature, mild acid conditions, on protein properties influence compared with
It is small.
Specific embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This.
Embodiment 1, a kind of highly selective chemical modification method for being directed to tryptophan and its residue, select aryl primary amine dyestuff
C.I. acid brown 4 is used as aryl primary amine dressing agent, and formaldehyde is as aldehyde component, tryptophan, tyrosine, histidine, cysteine, silk ammonia
Acid, threonine, arginine, asparatate, lysine, phenylalanine the higher amino acid of totally ten kinds of activity respectively as being repaiied
Jewelry.
Chemical modification method is specific as follows：
Select the formalin of 37% (quality %).
By C.I. acid browns 4, amino acid, formaldehyde (aqueous solution) in molar ratio 1：1：100 mixing, at this point, water and aryl primary amine
The mass ratio of dressing agent is about 50：1；Reacting liquid pH value is adjusted to 4.0 and (uses the sodium acetate and 16.4mL of 3.6mL 1mol/L
The acetic acid of 1mol/L is formulated, this is routine techniques), reaction temperature is 30 DEG C, soaking time 24 hours.
After reaction, pH value is adjusted to 5.0, and product is precipitated, is filtered, washed (in being washed with clear water and being to eluent
Property), dry.
Only when tryptophan is as object is modified, products therefrom is brown, that is, only tryptophan is used as and is modified object energy
Three component reaction products are enough obtained,
And other amino acid (confirm) with 4 reactionless phenomenon of C.I. acid browns through thin-layer chromatography and mass spectrography, illustrate the change
Learning method of modifying tryptophan has highly selective, and covalent bonding is formed between aryl primary amine dressing agent and tryptophan.
Comparative example 1-1, the use for cancelling formaldehyde in embodiment 1, remaining is equal to embodiment 1, the result of final gained
For：C.I. it is not chemically reacted between acid brown 4 and tryptophan.
Comparative example 1-2, by C.I. acid browns 4, amino acid, formaldehyde (aqueous solution) in molar ratio 1：1：2 are mixed；Remaining
It is equal to embodiment 1.Finally the result of gained is：C.I. it is not chemically reacted between acid brown 4 and tryptophan.
Embodiment 2, a kind of highly selective chemical modification method for being directed to tryptophan and its residue, select open-chain crown ether to make
For aryl primary amine dressing agent, aldehyde component selects acetaldehyde, polypeptide A (the amino acid sequence Tyr-Glu-Tyr- containing trp residue
Ala-Trp-Ser-Ser, wherein Trp are tryptophan, and Tyr is tyrosine, and Glu is glutamic acid, and Ala is alanine, and Ser is silk ammonia
Acid) as being modified object.
Chemical modification method is specific as follows：
0.007mmol polypeptide As and the open-chain crown ether of 0.05mmol (5.35mg) and the acetaldehyde of 0.15mmol；Reaction system
Middle water is 35mL；PH value is 4.5, and reaction temperature is 37 DEG C, soaking time 22 hours.
After reaction, through superheated water and cold water washing (washing 10h by 60 DEG C and 15 DEG C of deionized waters), so as to remove
It removes to adsorb the impurity on polypeptide, is filtered, washed (it is neutral to be washed with clear water to eluent), dries.Analysis result shows one
A open-chain crown ether molecule is connected to by C-N abutments on tryptophan and its residue, and modification rate reaches 75%, illustrates that the chemistry is repaiied
Decorations method equally has good reactivity to the tryptophan in polypeptide structure and its residue.
Explanation：Using the content of the unreacted open-chain crown ether of liquid chromatogram measuring, the open-chain crown ether pair calculated
The reactivity of tyrosine residue, as modification rate.
Embodiment 3, a kind of highly selective chemical modification method for being directed to tryptophan and its residue, select C.I. acid greens 20
As aryl primary amine dressing agent, aldehyde component selects pyroracemic aldehyde, and the mulberry silk for containing trp residue is used as and is modified object.
Chemical modification method is specific as follows：
The C.I. acid browns 4 of 0.35g Light of Mulberry Silk Fabrics (containing about 0.007mmol tryptophans) and 7.5mg (0.07mmol) and
The pyroracemic aldehyde mixing of 7.0mmol；Water is 15mL in reaction system；PH value is 5.0, and reaction temperature is 30 DEG C, and soaking time 20 is small
When.
After reaction, each 10h then is washed by 60 DEG C and 15 DEG C of deionized waters, so as to remove absorption on silk
Impurity dries.
Silk is dyed to brown after reaction, measures fiber strength loss late<5%.It strips method (100 DEG C, 20min) through DMF
It proves, the dyestuff of Covalent bonding together is formed with silk trp residue to be stripped down.
It will be through 3 the method for embodiment treated silk according to national standard《Color fastness to washing GB/T3921-2008》
And national standard《Colour fastness to rubbing experiment GB/T 3920-2008》.Fabric color depth K/S is 12.3 after dyed fabric is soaped, resistance to
Fastness to wet rubbing is 3-4 grades, and it is 4-5 grades that washing fastness, which is stained with cotton, and it is 4 grades to be stained with silk, and discoloration as former state is 4 grades, it is known that this method is stained with
Silk fabric have excellent color fastness.The above results show mild chemical modification condition to silk fiber physical mechanical
Performance is smaller, and forms stable covalent bonding between dressing agent (dyestuff) and protein (silk).
Comparative example 2-1：Cancel the use of pyroracemic aldehyde in above-described embodiment 3, remaining is equal to embodiment 3；The result of gained
For：Fabric color depth K/S is 1.2 after soaping, and dyed fabric wet rubbing fastness is 3 grades, and it is 3 grades that washing fastness, which is stained with cotton, and being stained with silk is
3-4 grades, discoloration as former state is 4 grades.
And after the present invention adds in formaldehyde, after soaping, fabric color depth K/S is 12.3, and dyed fabric wet rubbing fastness is 3-4
Grade, it is 4-5 grades that washing fastness, which is stained with cotton, and it is 4 grades to be stained with silk, and discoloration as former state is 4 grades.Not only color depth value increases, but also color fastness improves.
Comparative example 2-2, the dosage of pyroracemic aldehyde in embodiment 3 is made into 0.14mmol by 7.0mmol, remaining is equal to implementation
Example 3.The result of gained is：Fabric color depth K/S is 1.3 after soaping, and dyed fabric wet rubbing fastness is 3 grades, and washing fastness is stained with
Cotton is 3 grades, and it is 3-4 grades to be stained with silk, and discoloration as former state is 3-4 grades.
Finally, it should also be noted that it is listed above be only the present invention several specific embodiments.Obviously, this hair
Bright to be not limited to above example, acceptable there are many deform.Those of ordinary skill in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Claims (7)
1. for tryptophan and its highly selective chemical modification method of residue, it is characterized in that：
Using tryptophan or the substance for containing trp residue as being modified object, Yu Shuizhong, the aryl primary amine under the action of aldehyde component
Highly selective three components electrophilic substitution reaction occurs for dressing agent and the tryptophan/trp residue being modified in object, thus will be fragrant
Primary amine modification agent molecule is connected firmly to covalent bond on tryptophan/trp residue.
2. the highly selective chemical modification method according to claim 1 for being directed to tryptophan and its residue, it is characterized in that：
The molar ratio of the aryl primary amine dressing agent and tryptophan/trp residue is 0.1~10：1, aryl primary amine dressing agent and aldehyde group
The molar ratio divided is 1：3~100；The pH value for adjusting entire reaction system is 4.0~6.5, in 20~60 DEG C of insulation reactions 1~24
Hour；
It is washed rear dry to react products therefrom.
3. the highly selective chemical modification method according to claim 2 for being directed to tryptophan and its residue, it is characterized in that：Water
Mass ratio with aryl primary amine dressing agent is 20~7000：1.
4. the highly selective chemical modification method for being directed to tryptophan and its residue according to Claims 2 or 3, feature
It is：
At least need that there are an aryl primary amine segments in the structure of the aryl primary amine dressing agent.
5. the highly selective chemical modification method according to claim 4 for being directed to tryptophan and its residue, it is characterized in that institute
State aryl primary amine dressing agent for it is following any one：Aniline, naphthylamines, anil, naphthylamine derivative；
The anil is open-chain crown ether, acetparaminosalol aniline, P-nethoxyaniline, aryl primary amine dyestuff C.I. are acid
Palm fibre 4, C.I. acid greens 20.
It is 6. special according to any highly selective chemical modification method for being directed to tryptophan and its residue of claim 2~5
Sign is：
The aldehyde component is formaldehyde, acetaldehyde, propionic aldehyde, butyraldehyde, pyroracemic aldehyde, glutaraldehyde or benzaldehyde.
It is 7. special according to any highly selective chemical modification method for being directed to tryptophan and its residue of claim 2~5
Sign is：
Object is modified as tryptophan, the polypeptide A containing trp residue, the protein containing trp residue.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110592977A (en) * | 2019-08-20 | 2019-12-20 | 浙江理工大学上虞工业技术研究院有限公司 | Rapid reactive dyeing method suitable for silk |
| WO2020254606A1 (en) * | 2019-06-21 | 2020-12-24 | Ascendis Pharma A/S | Conjugates of heteroaromatic nitrogen-comprising compounds |
| CN114262356A (en) * | 2021-12-28 | 2022-04-01 | 深圳湾实验室坪山生物医药研发转化中心 | Method for modifying protein tryptophan residue |
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| CN101809032A (en) * | 2007-08-24 | 2010-08-18 | 诺沃-诺迪斯克有限公司 | Method for selectively modifying a protein |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020254606A1 (en) * | 2019-06-21 | 2020-12-24 | Ascendis Pharma A/S | Conjugates of heteroaromatic nitrogen-comprising compounds |
| CN110592977A (en) * | 2019-08-20 | 2019-12-20 | 浙江理工大学上虞工业技术研究院有限公司 | Rapid reactive dyeing method suitable for silk |
| CN110592977B (en) * | 2019-08-20 | 2021-10-26 | 浙江理工大学上虞工业技术研究院有限公司 | Rapid reactive dyeing method suitable for silk |
| CN114262356A (en) * | 2021-12-28 | 2022-04-01 | 深圳湾实验室坪山生物医药研发转化中心 | Method for modifying protein tryptophan residue |
| CN114262356B (en) * | 2021-12-28 | 2023-10-24 | 深圳湾实验室坪山生物医药研发转化中心 | Method for modifying tryptophan residues of protein |
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