CN117417292A - Heteroaryl-containing unnatural amino acid and application thereof - Google Patents
Heteroaryl-containing unnatural amino acid and application thereof Download PDFInfo
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- CN117417292A CN117417292A CN202310160246.4A CN202310160246A CN117417292A CN 117417292 A CN117417292 A CN 117417292A CN 202310160246 A CN202310160246 A CN 202310160246A CN 117417292 A CN117417292 A CN 117417292A
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- Prior art keywords
- amino acid
- unnatural amino
- alkyl
- group
- recombinant human
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- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000006011 modification reaction Methods 0.000 description 1
- 150000004682 monohydrates Chemical class 0.000 description 1
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 239000012299 nitrogen atmosphere Substances 0.000 description 1
- 201000008106 ocular cancer Diseases 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 238000006146 oximation reaction Methods 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
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- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 230000006916 protein interaction Effects 0.000 description 1
- 125000003373 pyrazinyl group Chemical group 0.000 description 1
- 125000002098 pyridazinyl group Chemical group 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 125000000714 pyrimidinyl group Chemical group 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
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- 238000012216 screening Methods 0.000 description 1
- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 239000012265 solid product Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- ZXUCBXRTRRIBSO-UHFFFAOYSA-L tetrabutylazanium;sulfate Chemical compound [O-]S([O-])(=O)=O.CCCC[N+](CCCC)(CCCC)CCCC.CCCC[N+](CCCC)(CCCC)CCCC ZXUCBXRTRRIBSO-UHFFFAOYSA-L 0.000 description 1
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 description 1
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Abstract
The invention provides a medicine containingAn unnatural amino acid having a heteroaryl group, which is a compound having a structure as shown in formula (I) or an enantiomer thereof. The invention also provides application of the unnatural amino acid. The invention also provides a recombinant human interleukin 2-polyethylene glycol conjugate and application thereof. The non-natural amino acid containing heteroaryl provided by the invention has higher protein expression level when preparing recombinant protein, and higher synthesis efficiency when forming conjugate, thus having better applicability, simple preparation process, no need of high cost and being suitable for large-scale production. The recombinant human interleukin 2-polyethylene glycol conjugate provided by the invention realizes the accurate fixed-point coupling of PEG and IL-2 by using the unnatural amino acid, thereby realizing the prolongation of the half-life of IL-2 in vivo and being beneficial to the immunotherapy of tumors,
Description
Technical Field
The invention relates to the field of biopharmaceuticals, in particular to a heteroaryl-containing unnatural amino acid and application of the unnatural amino acid.
Background
The natural amino acids composing the protein are only 20 kinds, the kinds of carried functional groups are limited, and the non-natural amino acids obtained after modification and transformation are more in kinds, and the carried functional groups are very various, so that various modification reactions such as click chemistry, photochemistry, glycosylation, fluorescent color development and the like can be carried out.
By introducing unnatural amino acids containing special groups into proteins, various scientific researches and product development applications can be realized, for example, photosensitive unnatural amino acids are introduced into proteins, or the unnatural amino acids are specially marked, so that the interaction between proteins can be conveniently researched; for example, the unnatural amino acid is utilized to carry out directional modification of the enzyme so as to improve the enzyme activity and the stability of the enzyme or facilitate the efficient immobilization of the enzyme; for another example, safe live bacterial or live viral vaccines can be prepared using the feature that unnatural amino acid insertion is not possible in conventional hosts. An important application of introducing unnatural amino acids into proteins by using codon extension technology is to perform site-directed modification on proteins, change the characteristics of functions, stability, half-life and the like of the proteins, and can be used for developing innovative biological drugs. It follows that unnatural amino acids have a very important role and a very broad range of applications.
Chinese patent ZL 202111019771.1 discloses an unnatural amino acid derived from lysine, which has a carbonyl group introduced at its end as an active reactive group, and further comprises an aryl group attached to the terminal carbonyl group. Compared with common terminal azido unnatural amino acids (such as Lys-azido), the unnatural amino acids are simpler and more convenient to prepare, have better safety, are not easy to inactivate when inserted into proteins, have higher binding rate with a coupling part, and have higher stability of the obtained conjugate, so that the conjugate can be used for preparing recombinant proteins, recombinant protein conjugates and the like.
ParA-Acetylphenylalanine (pAF) is also one of the currently common unnatural amino acids (e.g., chinese patent application CN 111212661 a), which can also be conveniently inserted into proteins and coupled to other substances (e.g., polymers, drugs, etc.).
The unnatural amino acids have good application potential in preparing recombinant proteins and recombinant protein conjugates. Therefore, the modified amino acid is further transformed or modified on the basis of the modified amino acid, and has important significance for expanding the variety and application of the unnatural amino acid and improving the performance of the unnatural amino acid.
Disclosure of Invention
An object of the present invention is to provide a heteroaryl group-containing unnatural amino acid having a nitrogen atom-containing heteroaryl group introduced into its structure, which can increase the protein expression level when inserted into a recombinant protein, and which can be synthesized with higher efficiency when forming a conjugate, thereby greatly improving its applicability, and its use.
It is another object of the present invention to provide a recombinant human interleukin 2-polyethylene glycol conjugate and uses thereof.
The invention provides a non-natural amino acid containing heteroaryl, which is characterized in that the non-natural amino acid is a compound with a structure shown as a formula (I) or an enantiomer thereof,
wherein ring A represents a substituted or unsubstituted 5-to 10-membered heteroaryl group containing 1, 2 or 3 nitrogen atoms;
l represents a substituted or unsubstituted C1-C20 linear or branched alkylene group, one or more of which-CH 2 -optionally replacingIs one or more of-O-, -NH-, -C (O) -, -S (O) -;
r represents a substituted or unsubstituted C0-C10 linear or branched alkylene group, one or more of which-CH 2 -optionally replaced by one or more of-O-, -NH-, -C (O) -;
when the rings A, L, R each independently represent a substituted group, the substituents are selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, hydroxy, mercapto, halogen, nitro, cyano, amino, -NH (C1-C6 alkyl), -N (C1-C6 alkyl) 2 -CO (C1-C6 alkyl), -COO (C1-C6 alkyl), -CONH 2 -CONH (C1-C6 alkyl), -CON (C1-C6 alkyl) 2 One or more of C3-C10 cycloalkyl, C3-C10 heterocyclic group and C6-C20 aryl.
In the unnatural amino acid provided by the invention, "C0-Cn" includes C0-C1, C0-C2 and … … C0-Cn, when C0 is represented, the group is absent, and the C atoms at the two ends are directly connected to form a bond. For example, the "C1-C20" group refers to having 1 to 20 carbon atoms in the moiety, and the "C0-C10" group refers to having 0 to 10 carbon atoms in the moiety. For example, the "C0-C6" group refers to a moiety having from 0 to 6 carbon atoms in the moiety, i.e., the group is absent, contains 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms. And so on.
The unnatural amino acids provided by the invention include optically pure enantiomers and racemates.
In some embodiments of the unnatural amino acid according to the invention, the unnatural amino acid is a compound having a structure as shown in formula (I-1) or formula (I-2) or an enantiomer thereof,
wherein the ring A, R is as defined in any one of the preceding claims;
x represents a substituted or unsubstituted C0-C10 linear or branched alkylene group, one of whichOne or more-CH 2 -optionally replaced by one or more of-O-, -NH-, -C (O) -;
when X represents a substituted group, the substituent is selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, hydroxy, mercapto, halogen, nitro, cyano, amino, -NH (C1-C6 alkyl), -N (C1-C6 alkyl) 2 -CO (C1-C6 alkyl), -COO (C1-C6 alkyl), -CONH 2 -CONH (C1-C6 alkyl), -CON (C1-C6 alkyl) 2 One or more of C3-C10 cycloalkyl, C3-C10 heterocyclic group and C6-C20 aryl.
In some preferred embodiments, the ring a represents a 5-6 membered heteroaryl group containing 1 or 2 nitrogen atoms, including but not limited to pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, and the like.
In some preferred embodiments, the ring a represents pyridinyl.
In some preferred embodiments, each of said R, X independently represents a C0-C6 linear or branched alkylene group, one or more of which-CH 2 -optionally replaced by-O-.
In some embodiments of the unnatural amino acid according to the invention, the unnatural amino acid is a compound having a structure as shown in formula (I-3) or formula (I-4) or an enantiomer thereof,
wherein the ring A, X is as defined in any one of the preceding claims.
In some preferred embodiments, when the rings A, X each independently represent a substituted group, the substituents are selected from one or more of halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, including, but not limited to F, cl, br, I, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, methoxy, trifluoromethyl, and the like.
In some embodiments of the unnatural amino acid according to the invention, the unnatural amino acid is a compound having a structure as shown in formula (I-5), formula (I-6) or formula (I-7) or an enantiomer thereof,
wherein Y represents a linear or branched alkylene group having 0 to 6 carbon atoms;
r' represents one or more of hydrogen, halogen, C1-C4 alkyl, C1-C4 alkoxy and C1-C4 haloalkyl;
n represents 0, 1, 2 or 3.
In some preferred embodiments, Y represents a C0-C4 linear alkylene group.
In some embodiments of the unnatural amino acid according to the invention, the unnatural amino acid is a compound with one of the following structures:
the invention also provides the application of the unnatural amino acid in any of the above technical schemes in preparing recombinant protein or recombinant protein conjugate.
In the use according to the present invention, the recombinant protein may be obtained by inserting the unnatural amino acid according to any of the above embodiments at any site and in any number into any kind of protein commonly known in the art. The recombinant protein conjugate may be a conjugate obtained by coupling any recombinant protein obtained with a coupling moiety common in the art, wherein the coupling moiety includes, but is not limited to, a polymer (e.g., polyethylene glycol of any molecular weight), a protein, a polypeptide, a small molecule drug, and the like.
In some preferred embodiments, the recombinant protein may be recombinant human interleukin 2, and/or the recombinant protein conjugate may be a recombinant human interleukin 2-polyethylene glycol conjugate.
In other preferred embodiments, the recombinant protein may be recombinant human growth hormone, and/or the recombinant protein conjugate may be a recombinant human growth hormone-polyethylene glycol conjugate.
The invention also provides a recombinant protein, wherein at least one site in the amino acid sequence of the recombinant protein is the unnatural amino acid according to any one of the technical schemes.
Further, the recombinant protein may have a structure as shown in formula (II),
in the formula (II), D represents the residue of the unnatural amino acid according to any one of the above technical schemes, from which the aminocarboxylic acid moiety is removed, P 1 、P 2 Representing the linking moiety of the amino and carboxyl groups of the unnatural amino acid in the amino acid sequence, respectively.
The recombinant protein provided by the invention can be prepared by using a preparation method common in the field, and comprises cloning and expression of the recombinant protein containing unnatural amino acid by using a gene codon extension technology.
The recombinant protein provided by the invention can be obtained by inserting the unnatural amino acid into any kind of protein common in the field at any site and in any number, such as recombinant human growth hormone, recombinant human interleukin 2 and the like.
The invention also provides a recombinant protein conjugate, wherein the end carbonyl group of the unnatural amino acid in the recombinant protein contains NH 2 The coupling moiety of the-O- "end group forms an oxime bond.
Further, the recombinant protein conjugate may have a structure as shown in formula (III),
in formula (III), D' represents the residue of the recombinant protein according to any one of the preceding technical schemes, from which the terminal carbonyl moiety of the unnatural amino acid is removed, D "represents the removal of the coupling moiety of" NH 2 -O- "residues of the end group.
In the recombinant protein conjugates provided by the invention, the coupling moiety can include polymers (e.g., polyethylene glycol of any molecular weight), proteins, polypeptides, small molecule drugs, and the like. The different kinds of coupling moieties can be coupled to the recombinant protein alone or after the different kinds of coupling moieties form a linkage.
In some preferred embodiments, the recombinant protein may be recombinant human growth hormone, recombinant human interleukin 2, or the like, the recombinant protein containing "NH" 2 The coupling moiety of the-O- "end group may be a moiety containing an" NH 2 -O- "terminal polyethylene glycol.
In some preferred embodiments, an "NH" is contained 2 -O- "terminal polyethylene glycol has the following structural formula:
wherein, contains "NH 2 The molecular weight of the polyethylene glycol of the-O- "end group may be from 10 to 100KD, including but not limited to molecular weight ranges of about 10KD, 20KD, 30KD, 40KD, 50KD, 60KD, 70KD, 80KD, 90KD, 100KD, etc., or any combination thereof. Preferably, it contains "NH 2 The molecular weight of the polyethylene glycol of the-O- "end groups may be from 20 to 50KD.
The invention also provides a recombinant human interleukin 2-polyethylene glycol conjugate, which comprises recombinant human interleukin 2 containing at least one unnatural amino acid and PEG conjugated to the at least one unnatural amino acid, wherein the unnatural amino acid is as set forth in any one of the preceding technical schemesThrough the end carbonyl groups of said unnatural amino acid with a catalyst comprising an amino acid sequence of "NH" 2 -O- "the PEG of the end group forms an oxime bond to couple the PEG to the at least one unnatural amino acid.
In some preferred embodiments, the recombinant human interleukin 2 is a protein shown in SEQ ID NO. 3 or a functionally active fragment thereof.
In some more preferred embodiments, the recombinant human interleukin 2 comprising at least one unnatural amino acid is selected from the group consisting of one or more positions corresponding to position P34, position K35, position T37, position R38, position L40, position T41, position F42, position K43, position F44, position Y45, position E61, position E62, position K64, position P65, position E67, position E68, position N71, position L72, and position Y107 of SEQ ID NO. 2.
In some preferred embodiments, the catalyst contains "NH 2 The molecular weight of the PEG of the-O- "end group is 20-50 KD.
The preparation and purification method of the recombinant human interleukin 2-polyethylene glycol conjugate provided by the invention can refer to the method described in Chinese patent ZL 202111019771.1.
The invention also provides the use of the recombinant human interleukin 2-polyethylene glycol conjugate according to any one of the previous technical schemes in preparing a medicament for promoting the immune function of interleukin 2.
The invention also provides the use of the recombinant human interleukin 2-polyethylene glycol conjugate according to any one of the previous technical schemes in preparing a medicament for treating cancer.
The invention also provides the preparation of the recombinant human interleukin 2-polyethylene glycol conjugate for expanding CD8 + Use of T cells in medicine.
In some preferred embodiments, the cancer includes, but is not limited to, renal cell carcinoma, liver cancer, rectal cancer, lymphatic cancer, bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, and the like.
The technical scheme provided by the invention has the following advantages:
(1) The unnatural amino acid provided by the invention has the advantages that the end-group carbonyl and the nitrogen-containing heteroaryl connected with the end-group carbonyl are introduced into the structure of the unnatural amino acid, the end-group carbonyl can conveniently form a conjugate, and the heteroaryl can further enhance the stability of the obtained conjugate. In addition, compared with the unnatural amino acid with aryl at the same position, the unnatural amino acid provided by the invention has higher protein expression level when preparing recombinant protein, higher synthesis efficiency when forming conjugates, higher solubility and easier operation in fermentation broth and feeding process. Therefore, the unnatural amino acid provided by the invention has better applicability.
(2) The unnatural amino acid provided by the invention expands the variety of amino acid, so that the unnatural amino acid can be used as an amino acid derivative to be applied to various fields, especially to the preparation of recombinant protein or recombinant protein conjugate, and also expands the variety and application range of the recombinant protein or recombinant protein conjugate.
(3) The preparation process of the unnatural amino acid provided by the invention is simple and convenient, does not need high cost, and is suitable for large-scale production.
(4) The recombinant human interleukin 2-polyethylene glycol conjugate provided by the invention realizes the accurate fixed-point coupling of PEG and IL-2 by using the unnatural amino acid containing heteroaryl, thereby realizing the extension of the half-life of IL-2 in vivo. The recombinant human interleukin 2-polyethylene glycol conjugate provided by the invention can reduce the binding activity of IL-2 Ralpha and keep the binding activity of IL-2 Rbeta and IL-2 Rgamma relatively unchanged, thereby being capable of specifically promoting CD8 in tumor microenvironment + Proliferation of T cells, but for CD4 + Proliferation of T cells has no obvious effect, thereby facilitating immunotherapy of tumors.
Drawings
FIG. 1 is a schematic representation of the expression plasmid pET21-rhIL2T41 in example 4.
FIG. 2 is an SDS-PAGE electrophoresis of fermentation products obtained by adding different kinds of unnatural amino acids to rhIL-2-expressing strains in example 5.
FIG. 3A is an RP-HPLC chart of PEG-rhIL2-pAF5 and PEG-rhIL2-pAF5 in example 6.
FIG. 3B is a RP-HPLC chart of PEG-rhIL2-NBOK and PEG-rhIL2-NBOK3 in example 6.
FIG. 3C is an RP-HPLC chart of PEG-rhIL2-NPAK and PEG-rhIL2-NPAK2 in example 6.
Detailed Description
The technical scheme of the invention is further described in detail below with reference to specific embodiments.
The reagents or raw materials used in the examples of the present invention are all commercially available products unless otherwise specified; the experimental methods used, unless otherwise specified, are conventional in the art.
The unnatural amino acids NBOK and NPAK used in the examples of the present invention have the following structural formulas, respectively, and are self-made with reference to Chinese patent ZL 202110863816.7.
NPAK：
NBOK：
The percentages used in the examples of the present invention are mass percentages unless otherwise indicated and the fractions relating to the eluent are volume ratios.
Abbreviations used in the examples of the present invention represent the following meanings:
example 1 preparation of unnatural amino acid pAF5
The structural formula of pAF5 is shown below:
the synthesis process is as follows:
the method comprises the following steps:
a) In the reaction flask, 2-acetyl-5-methylpyridine (1.0 g,7.4 mmol) was added and solvent DCE (20.0 mL) was added. NBS (1.58 g,8.9 mmol) and BPO (0.09 g,0.37 mmol) were added. After the mixture was refluxed at 95℃for 24 hours, the vessel was cooled in ice water to precipitate a solid. The solid was removed by filtration, using saturated Na 2 HCO 3 Washing 3 times. Extraction 3 times with DCM, combining the organic phases, drying over anhydrous sodium sulphate, filtration, concentration under reduced pressure and purification by column chromatography (eluent: PE: ea=40:1) gives product 1-1 (0.95 g, 60% yield).
b) In the reaction flask, product 1-1 (0.95 g,4.4 mmol) was added and ethyl diphenylmethylene glycine (1.17 g,4.4 mmol) was added. Tetra-n-butyl ammonium sulfate (n-BuNHSO) 4 1.5g,4.4 mmol) and sodium hydroxide (0.42 g,10.56 mmol) were first prepared as a solution with 10mL of water and 10mL of methylene chloride, and then the solution was slowly dropped into the reaction flask. Stir at room temperature for 18 hours. The extracts were extracted 3 times with DCM and the organic phases combined. Concentrated under reduced pressure and purified by column chromatography (eluent: PE: ea=20:1) to give product 1-2 (0.84 g, yield 41%).
c) In the reaction flask, product 1-2 (0.84 g,2.1 mmol) was added followed by 2M hydrochloric acid (formulated with 2mL concentrated hydrochloric acid and 10 mL). The mixture was refluxed at 75 ℃ for 12 hours. Washing 3 times with ethyl acetate, the aqueous phase was lyophilized in a freeze-dryer to give the hydrochloride salt of the brown solid product pAF5 (0.5 g, 97% yield).
1 H-NMR (400 MHz, heavy water): δ8.93 (d, j=1.8 hz, 1H), 8.75 (dd, j=8.2, 2.0hz, 1H), 8.61 (d, j=8.4 hz, 1H), 4.49 (dd, j=7.6, 6.4hz, 1H), 3.64-3.56 (m, 2H), 2.81 (s, 3H), 2.06 (s, 1H).
EXAMPLE 2 preparation of unnatural amino acid NBOK3
The structural formula of NBOK3 is shown below:
the synthesis process is as follows:
the method comprises the following steps:
a) In a reaction flask, 2-acetyl-5-methylpyridine (4.30 g,33.3 mmol) was added, solvent DCE (80 mL) was added, NBS (10.10 g,56.6 mmol) and BPO (0.41 g,1.7 mmol) were added, the mixture was refluxed at 85℃for 24 hours, then the vessel was cooled in ice water, the solid was precipitated, and the solid was removed by filtration using saturated Na 2 CO 3 Washing for 3 times, adding anhydrous sodium sulfate for drying, filtering, and concentrating under reduced pressure to obtain a crude product 2-1, wherein the crude product is directly used in the next step without purification.
b) In a reaction flask, product 2-1 was added, solvent dioxane (50 mL) and water (50 mL) were added, and further calcium carbonate (20.0 g,120 mmol) was added, and the mixture was refluxed at 100 ℃ for 24 hours, cooled to room temperature, filtered to remove solids, distilled under reduced pressure to remove excess dioxane, extracted 3 times with ethyl acetate, the organic phases were combined, concentrated under reduced pressure, purified by column chromatography (eluent: PE: ea=2:1) to give product 2-2 (1.8 g, 36% yield in two steps).
c) In a two-necked reaction flask, p-nitrophenyl chloroformate (3.20 g,14.4 mmol) was added, solvent DCM (50.0 mL) was added, the temperature was lowered to 0℃and product 2-2 (1.80 g,12 mmol) and pyridine (1.2 mL,14.4 mmol) were added, and after stirring at room temperature for 18 hours, the mixture was washed with water 2 times, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (eluent: PE: ea=5:1 and DCM) to give product 2-3 (2.91 g, 77% yield).
d) In a reaction flask, product 2-3 (2.91 g,9.2 mmol) and Fmoc-Lys-OH hydrochloride (3.70 g,9.2 mmol) were added, solvent dioxane (25 mL) and water (25 mL) were added, triethylamine (3.2 mL,23 mmol) was further added, after the mixture was reacted at room temperature for 18 hours, an appropriate amount of 1M HCl solution was added, extracted with ethyl acetate, concentrated under reduced pressure, and purified by column chromatography (eluent: DCM: meOH: acoh=20:1:0.5) to afford product 2-4 (4.69 g, 94% yield).
e) In a reaction flask, product 2-4 (4.69 g,8.6 mmol) was taken and dissolved in DCM (20 mL), diethylamine (10 mL) was added and reacted at room temperature for 6 hours, the product precipitated, filtered and slurried 3 times with DCM to give the product NBOK3 as a pale green solid (1.60 g, yield 58%).
1 H-NMR (400 MHz, heavy water): delta 8.93 (s, 1H), 8.74 (t, j=8.0 hz, 1H), 8.63 (d, j=8.2 hz, 1H), 5.37 (s, 2H), 4.07 (t, j=6.4 hz, 1H), 3.15 (t, j=6.4 hz, 2H), 2.82 (s, 3H), 2.01-1.91 (m, 2H), 1.59-1.53 (m, 2H), 1.49-1.39 (m, 2H).
EXAMPLE 3 preparation of unnatural amino acid NPAK2
The structural formula of NPAK2 is shown below:
the synthesis process is as follows:
the method comprises the following steps:
a) Into a reaction flask, 2-acetyl-5-bromopyridine (5.0 g,25.0 mmol) was charged, and diethyl malonate (28.0 g,175 mmol) and KHCO were added under nitrogen atmosphere 3 (3.80 g,37.5 mmol) and K 2 CO 3 (5.20 g,37.5 mmol) and then Pd (dba) were added 2 (0.144 g,0.25 mmol) and P (t-Bu) 3 HBF 4 (0.159 g,0.55 mmol) and after the addition was completed, nitrogen protection was replaced, and the temperature was raised to 160℃for 24 hours. After completion of the TLC detection, water (150 mL) was added to the reaction solution, the organic phases were combined, washed 2 times with water, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification by column chromatography (eluent: PE: ea=4:1) afforded product 3-1 (1.73 g, 34% yield) as a yellow solid.
b) In a reaction flask, liOH monohydrate (1.05 g,25.0 mmol) and water (10 mL) were added, ethanol (20 mL) was added, the product 3-1 (1.73 g,8.35 mmol) was added, and after stirring at room temperature for 2 hours, TLC detection was complete, and after adjusting pH=1-2 by adding 2M HCl solution to the reaction solution, freeze-drying was performed with a freeze dryer to obtain the crude product 3-2. Without purification, it was used directly in the next step.
c) In the reaction flask, product 3-2 (calculated as 8.35 mmol) was added, NHS (1.4 g,12.53 mmol), EDCI (2.4 g,12.53 mmol), acetonitrile (50 mL) was added. After stirring at room temperature for 24 hours and completion of TLC detection, concentration under reduced pressure and purification by column chromatography (eluent: PE: EA=1:1) gave product 3-3 (0.61 g, two-step yield 27%).
d) In a reaction flask, product 3-3 (0.61 g,2.2 mmol) was added, triethylamine (1.8 mL,5.5 mmol), fmoc-Lys-OH hydrochloride (2.1 g,5.5 mmol), dioxane (20 mL) and water (20 mL) were added sequentially and the reaction was stirred at room temperature for 18 hours. After completion of TLC detection, the reaction was concentrated under reduced pressure, extracted 3 times with EA and dried over anhydrous sodium sulfate to give the crude product 3-4. Without purification, it was used directly in the next step.
e) In the reaction flask, product 3-4, DCM (20 mL) and diethylamine (10 mL) were added and stirred at room temperature for 6 hours. After completion of the TLC detection reaction, the reaction mixture was concentrated under reduced pressure. The solution was stirred after adding 2M hydrochloric acid. Lyophilization was performed on a lyophilizer and the resulting solid was slurried with ethyl acetate and a little ethanol to give NPAK2 hydrochloride as a pale yellow solid (181 mg, overall yield 2.1%).
1 H-NMR (400 MHz, heavy water): delta 8.54 (d, j=1.6 hz, 1H), 8.04 (d, j=8.0 hz, 1H), 7.92 (dd, j=8.2, 2.0hz, 1H), 3.74 (s, 2H), 3.67 (t, j=6.8 hz, 1H), 3.23 (t, j=7.2 hz, 2H), 2.70 (s, 3H), 1.88-1.81 (m, 2H), 1.62-1.53 (m, 2H), 1.45-1.36 (m, 2H).
EXAMPLE 4 construction of recombinant interleukin 2 (IL-2) expressing Strain containing unnatural amino acids
1) Acquisition of helper plasmids: helper plasmids pEVOL-pAcFRS.2.t1 (accession # 73544) and pSupAR-MbpyleS (accession # 91705) are from the plasmid deposit organization Addgene. pEVOL-pAcFRS.2.t1 can express tRNA and tRNA synthetases that specifically recognize p-Acetylphenylalanine (pAF) and pAF5 in E.coli; pSupAR-MbPylRS can express tRNAs and tRNA synthetases in E.coli that specifically recognize NPAK, NBOK, NPAK and NBOK3. The helper plasmid is obtained by shaking culture in LB medium added with 37.5mg/L chloramphenicol.
2) Construction of recombinant interleukin 2 expression plasmid pET21-rhIL2T41 containing a stop codon TAG inside the reading frame: the coding gene amino acid sequence of the homo sapiens interleukin 2 (amino acid sequence shown as SEQ ID NO: 1) is obtained from the national center database of biological information, and the signal peptide sequence consisting of 20 amino acids at the N-terminal is removed (which is excised in the process of processing and maturation of IL-2 protein molecules) to obtain the sequence SEQ ID NO:2.
Mutation of the 125 th cysteine codon to serine codon (the 125 th cysteine is not involved in disulfide bond formation, but rather interferes with the formation of normal disulfide bond during renaturation of the recombinant human IL-2 protein inclusion body, and the renaturation efficiency can be improved without significantly affecting the activity thereof after mutation), and in order to express recombinant proteins in E.coli, it is necessary to add methionine Met at the N-terminus of the protein sequence for initiating translation of the protein, thereby obtaining the mature recombinant human IL-2 protein sequence SEQ ID NO:3.
The codon of threonine at position 41 of SEQ ID NO. 3 is changed into an amber codon (TAG) for inserting unnatural amino acid, and finally the amino acid sequence of recombinant human IL-2 is obtained (X represents unnatural amino acid, SEQ ID NO. 4). And synthesizing the complete DNA sequence by a total gene synthesis mode to obtain a gene sequence (SEQ ID NO: 5) of the recombinant human IL-2.
SEQ ID NO:1：
MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT
SEQ ID NO:2
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT
SEQ ID NO:3
MAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT
SEQ ID NO:4：
MAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLXFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT
SEQ ID NO:5：
CATATGGCGCCTACATCCAGCTCGACCAAAAAGACTCAACTGCAACTGGAACACCTGCTCCTGGATCTGCAAATGATTCTTAACGGTATCAATAACTACAAAAATCCGAAACTGACCCGTATGCTGTAGTTTAAATTCTATATGCCAAAGAAAGCGACCGAGCTGAAACATCTGCAGTGCCTGGAAGAGGAACTGAAACCGCTGGAGGAAGTTTTGAACCTGGCTCAGTCTAAAAACTTTCACCTGCGCCCTCGTGACCTGATTTCCAATATCAACGTGATTGTTCTGGAACTGAAAGGCTCTGAAACCACGTTTATGTGCGAGTACGCCGATGAAACCGCCACGATTGTGGAATTTCTGAATCGCTGGATCACCTTCAGCCAGAGCATTATTAGCACGCTGACCTAATGACTCGAG
The gene sequence (SEQ ID NO: 5) of recombinant human IL-2 was cloned between Nde I and Xho I cleavage sites of pET21b (Novagen, cat# 69741-3) by one-step subcloning to obtain expression plasmid pET21-rhIL2T41, which was confirmed to be identical to the expected sequence by sequencing. pET21-rhIL2T41 can be used to express recombinant human IL-2 in which the amino acid codon at position 41 has been replaced with an amber codon. The recombinant human IL-2 expression plasmid pET21-rhIL2T41 is shown in figure 1.
3) Construction of pAF or pAF 5-containing recombinant IL-2-expressing strains: the helper plasmid pEVOL-pAcFRS.2.T1 and the expression plasmid pET21-rhIL2T41 were co-transformed into competent cells of E.coli Origamine B (DE 3) (Novagen, cat# 70911-3), and screened using LB medium containing 100mg/L ampicillin and 37.5mg/L chloramphenicol to obtain a double resistant strain, i.e., an expression strain of recombinant human IL-2, designated "IL2 (pAF/pAF 5) -OB".
4) Construction of recombinant IL-2 expressing strains containing NPAK, NBOK, NPAK2 or NBOK3: the helper plasmid pSupAR-MbPylRS and the expression plasmid pET21-rhIL2T41 were co-transformed into competent cells of E.coli OrigaminoB (DE 3) (Novagen, cat# 70911-3), and screened using LB medium containing 100mg/L ampicillin and 37.5mg/L chloramphenicol to obtain a double resistant strain, i.e., an expression strain of recombinant human IL2, designated "IL2 (NPAK/NBOK/NPAK-2/NBOK-3) -OB".
EXAMPLE 5 fermentative expression and purification of recombinant human IL-2 containing unnatural amino acids
1) Preparing a non-natural amino acid mother solution: pAF5, NBOK3 and NPAK2 obtained in examples 1 to 3 were dissolved in a 2 XYT medium (yeast extract 10g/L, tryptone 16g/L and NaCl 5 g/L) to prepare a mother solution having a final concentration of 100mM, and sterilized at high temperature for use. Dissolving pAF and NPAK in 2 XYT culture medium to obtain mother solution with final concentration of 80mM (which is affected by solubility and is difficult to reach higher concentration), and sterilizing at high temperature; NBOK is dissolved after being heated by a 2 XYT culture medium to prepare a mother solution with a final concentration of 10mM (which is affected by the solubility and is difficult to reach higher concentration), and the mother solution is sterilized at high temperature for standby.
2) The IL2 (pAF/pAF 5) -OB expression strain obtained in example 4 was inoculated into 3 parts of a 2 XYT medium (yeast extract 10g/L, tryptone 16g/L, naCl 5g/L, 100mg/L ampicillin and 37.5mg/L chloramphenicol), and cultured at 37℃to give a bacterial liquid OD 600 To 3 parts of the bacterial liquid, IPTG (final concentration: 1 mM) and arabinose (final concentration: 0.2%) were added, respectively, at 2.0.+ -. 0.2%. To 2 parts of each of the corresponding unnatural amino acids pAF and pAF5 (final concentration: 1 mM) were added, and the remaining 1 part of IL2-OB bacterial solution was used as a negative control without unnatural amino acid.
3) IL2 (NPAK/NBOK/NPAK 2/NBOK 3) -OB expression strain obtained in example 4 was inoculated into 5 parts of 2 XYT medium (yeast extract 10g/L, tryptone 16g/L, naCl 5g/L, 100mg/L ampicillin and 37.5mg/L chloramphenicol), and cultured at 37℃to give a bacterial liquid OD 600 To 5 parts of the bacterial liquid, IPTG (final concentration: 1 mM) and arabinose (final concentration: 0.2%) were added, respectively, at 2.0.+ -. 0.2%. Wherein 1 part of IL2-OB bacterial solution is used as a negative control without adding unnatural amino acid, and the corresponding unnatural amino acid NPAK, NBOK, NPAK and NBOK3 (the final concentration is 1 mM) are added in the other 4 parts respectively.
4) Culturing the bacterial liquid at 37 ℃ for induction expression for 5-6 hours, taking 1mL of each culture liquid, and centrifuging at 10000rpm for 1min to collect bacterial bodies. The cells were resuspended to OD with PBS 600 Each strain was subjected to SDS-PAGE for 10, and the SDS-PAGE for each strain is shown in FIG. 2. The results of FIG. 2 show that the expression strain can express the target under the condition of adding 6 unnatural amino acids respectivelyThe protein contains pAF5 and NPAK2, the expression quantity of IL-2 is about 3g/L, and the expression quantity of IL-2 containing NBOK3 is about 2.5g/L; correspondingly, the expression level of IL-2 containing pAF and NPAK is about 1g/L, and the expression level of IL-2 containing NBOK is about 1.5g/L. Therefore, the expression quantity of the IL-2 containing pyridine ring unnatural amino acid is obviously larger than that of the IL-2 containing benzene ring unnatural amino acid.
5) The collected thalli are respectively resuspended by buffer (25mM Tris,6mM EDTA,1mM DTT,pH8.0), 1% DNase (1 mg/mL) is added, evenly mixed, and homogenized for 3 times by an ultrahigh pressure homogenizer under the pressure of 50-80 MPa. Centrifuging the homogenized solution at 10000rpm for 20min, and collecting the lower inclusion body coarse body. The obtained inclusion bodies were roughly washed twice with a washing buffer (20 mM Tris-HCl,100mM NaCl,2% Triton X-100, pH 8.0) and then washed once with ultrapure water to obtain purified inclusion bodies. The purified inclusion bodies were dissolved in denaturation buffer (20 mM Tris-HCl,100mM NaCl,6M guanidine hydrochloride, 1mM DTT, pH 8.0), centrifuged at 10000rpm after 30min, and the supernatant was collected as a denatured protein solution. Adding 4 times of renaturation buffer (20 mM Tris-HCl,100mM NaCl,pH8.0) into the collected denatured protein solution, fully stirring, standing for 12h, centrifuging at 10000rpm, and collecting the supernatant to obtain the renaturation protein solution. The renaturated protein solution is concentrated to 1/4 of the original volume by an ultrafiltration membrane bag (Millipore, biomax-5) with the molecular weight cut-off of 5kDa, the replacement buffer (20 mM citric acid-sodium citrate buffer, pH 4.0) is used for replacing the solution, the concentration of the protein is further concentrated to about 2.0mg/mL, the supernatant is collected after centrifugation at 10000rpm, and 1mL of the supernatant is packaged and stored at-80 ℃ to obtain rhIL2-pAF, rhIL2-pAF5, rhIL2-NPAK, rhIL2-NBOK, rhIL2-NPAK2 and rhIL2-NBOK3.
EXAMPLE 6 coupling reaction and purification of recombinant IL-2 containing unnatural amino acids
Synthetic route A
Synthetic route B
Synthesis of route C
Synthesis of route D
Synthesis of route E
Synthesis of route F
Site-directed coupling of PEG to rhIL-2 with site-directed mutagenesis inserted into pAF5, NBOK3, NPAK2 is shown in synthetic schemes A-C (wherein P 1 To P 2 The direction of (a) is the N-terminal to C-terminal direction of the amino acid sequence). Accordingly, site-directed coupling of PEG to rhIL-2 with site-directed mutagenesis insertion pAF, NBOK, NPAK is shown in synthetic schemes D-F (wherein P 1 To P 2 The direction of (a) is the N-terminal to C-terminal direction of the amino acid sequence).
Taking 30KD aminooxy PEG (i.e. hydroxylamine group PEG) oximation reaction for coupling rhIL-2 as an example, the coupling reaction is operated as follows: prior to the coupling reaction, the protein of interest obtained above was adjusted to 2.0mg/ml with a citrate buffer at pH4.0, 20mM, according to 1:15 (molar ratio, protein: aminooxy PEG) 30KD aminooxy PEG solid (purchased from Beijing Kenka technologies Co., ltd.) was added, and the mixture was dissolved by shaking sufficiently to give a clear and transparent solution, and then the reaction solution was sealed and reacted by shaking in a constant temperature shaking table (25 ℃ C., 100 rpm). After 48h the coupling was analyzed using RP-HPLC as shown in FIGS. 3A-3C.
The results show that the PEG coupling is realized by the rhIL-2 containing the benzene ring unnatural amino acid and the rhIL-2 containing the pyridine ring unnatural amino acid, and the coupling rates (100% -the concentration of the rhIL-2 remained after the coupling is finished/the concentration of the rhIL-2 at the time of the coupling reaction is multiplied by 100%) of each molecule are respectively as follows: PEG-rhIL2-pAF:85.2%, PEG-rhIL2-pAF5:91.0%, PEG-rhIL2-NBOK:86.4%, PEG-rhIL2-NBOK3:90.7%, PEG-rhIL2-NPAK:94.3%, PEG-rhIL2-NPAK2:98.5%. Therefore, the coupling rate of rhIL-2 containing pyridine ring unnatural amino acid is obviously higher than that of rhIL-2 containing benzene ring unnatural amino acid.
RP-HPLC analysis conditions were as follows:
mobile phase a (0.1% TFA-H) 2 O)；
Mobile phase B (0.1% TFA-ACN).
The coupled PEG-rhIL2 is respectively regulated to pH 3.0+/-0.2 by using an equilibrium buffer solution (20 mM sodium citrate buffer solution (pH=3.0)), the conductivity is less than or equal to 5ms/cm, the sample is loaded to Capto MMC, the linear elution (0-100% of eluent, 20 CV) is carried out by using an elution buffer solution (20 mM sodium citrate buffer solution-1M NaCl (pH=7.8)) and the target protein component is collected, thus the target protein sample with the purity of about 95% can be obtained.
Example 7 in vitro Activity assay of PEG-coupled recombinant IL-2 (PEG-rhIL 2)
The method adopts two cell lines, wherein the mouse CTLL-2 cell is a cell line containing IL-2Ra beta gamma, the human YT cell is a cell line containing IL-2 Rbeta gamma, and the PEG-rhIL2 activates a JAK-STAT signal path through combining with the IL-2R on the cell surface. The lower the variation percentage of the EC50 ratio of YT cells/CTLL-2 cells is, the better the effect of the sample on promoting immune function is; conversely, the better the effect of suppressing immune function.
The specific process is as follows: mouse CTLL-2 cells (from American Type Cul ture Collection) and human YT cells were cultured in respective media (CTLL-2 cell culture medium: RPMI 1640+10% FBS+400IU/mL rhIL-2, 2mM L-glutamine, 1mM sodium pyruvate; YT cell culture medium: RPMI 1+10% FBS+1mM Non-Essential Amino Acids Solution (from Gibco, cat. No. 11140050)) at 37℃under 5% carbon dioxide conditions to a sufficient amount, starved for 4 hours before detection, thenCell density was adjusted to 1X 10 6 cell/mL was kept ready. Different PEG-coupled recombinant rhIL-2 and rhIL-2 references (purchased from Beijing Soy Corp.S. Co., ltd., product number: P00020) were respectively subjected to gradient dilution, 6 concentrations of each sample (in CTLL-2 cell experiments, the concentration range of the reference was 0.004-4 ng/mL, 4-fold gradient dilution, in YT cell experiments, the concentration range of the reference was 2.1-510 ng/mL, 3-fold gradient dilution, and the concentration ranges of the other samples were obtained by pre-experimental screening and corresponded to the corresponding EC50 values in Table 1), cells were stimulated for 10 minutes at 37℃and then lysed, westernblot experiments were performed, hybridization was performed using pSTAT5 antibodies (purchased from CST, product catalog number: 9359L) and betA-Actin (purchased from CST, product catalog number: 8457S), and the amounts of pSTAT5 and betA-Actin proteins in cell lysates were detected, and gray scale 50 was calculated from pSTAT 5/betA-Actin results and sample concentrations. The results are shown in Table 1. The result shows that the EC50 value of rhIL-2 inserted with the unnatural amino acid containing pyridine ring and coupled with 30KD PEG at the T41 site is equivalent to that of rhIL-2 inserted with the unnatural amino acid containing benzene ring and coupled with 30KD PEG.
TABLE 1 STAT5 phosphorylation test results
The calculation formula is as follows: [30KD PEG-rhIL2 sample YT cell EC50/30KD PEG-rhIL-2 sample CTLL-2 cell EC50]/[ reference YT cell EC 50/reference CTLL-2 cell EC50 ]. Times.100%
Unless otherwise defined, all terms used herein are intended to have the meanings commonly understood by those skilled in the art.
The described embodiments of the present invention are intended to be illustrative only and not to limit the scope of the invention, and various other alternatives, modifications, and improvements may be made by those skilled in the art within the scope of the invention, and therefore the invention is not limited to the above embodiments but only by the claims.
Claims (10)
1. A heteroaryl-containing unnatural amino acid characterized by being a compound having the structure shown in formula (I) or an enantiomer thereof,
wherein ring A represents a substituted or unsubstituted 5-to 10-membered heteroaryl group containing 1, 2 or 3 nitrogen atoms;
l represents a substituted or unsubstituted C1-C20 linear or branched alkylene group, one or more of which-CH 2 -optionally replaced with one or more of-O-, -NH-, -C (O) -, -S (O) -;
r represents a substituted or unsubstituted C0-C10 linear or branched alkylene group, one or more of which-CH 2 -optionally replaced by one or more of-O-, -NH-, -C (O) -;
when the rings A, L, R each independently represent a substituted group, the substituents are selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, hydroxy, mercapto, halogen, nitro, cyano, amino, -NH (C1-C6 alkyl), -N (C1-C6 alkyl) 2 -CO (C1-C6 alkyl), -COO (C1-C6 alkyl), -CONH 2 -CONH (C1-C6 alkyl), -CON (C1-C6 alkyl) 2 One or more of C3-C10 cycloalkyl, C3-C10 heterocyclic group and C6-C20 aryl.
2. The unnatural amino acid according to claim 1, wherein the unnatural amino acid is a compound having a structure represented by formula (I-1) or formula (I-2) or an enantiomer thereof,
wherein the ring A, R is as defined in claim 1;
x represents a substituted or unsubstituted C0-C10 linear or branched alkylene group, one or more of which-CH 2 -optionally replaced by one or more of-O-, -NH-, -C (O) -;
when X represents a substituted group, the substituent is selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, hydroxy, mercapto, halogen, nitro, cyano, amino, -NH (C1-C6 alkyl), -N (C1-C6 alkyl) 2 -CO (C1-C6 alkyl), -COO (C1-C6 alkyl), -CONH 2 -CONH (C1-C6 alkyl), -CON (C1-C6 alkyl) 2 One or more of C3-C10 cycloalkyl, C3-C10 heterocyclyl, C6-C20 aryl;
preferably, the ring a represents a 5-to 6-membered heteroaryl group containing 1 or 2 nitrogen atoms, more preferably a pyridinyl group;
preferably, each of said R, X independently represents a C0-C6 linear or branched alkylene group, one or more of which-CH 2 -optionally replaced by-O-.
3. The unnatural amino acid according to claim 2, wherein the unnatural amino acid is a compound having a structure represented by formula (I-3) or formula (I-4) or an enantiomer thereof,
wherein the ring A, X is as defined in claim 2;
preferably, when the rings A, X each independently represent a substituted group, the substituents are selected from one or more of halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl.
4. The unnatural amino acid according to claim 1 to 3, wherein the unnatural amino acid is a compound having a structure represented by formula (I-5), formula (I-6) or formula (I-7) or an enantiomer thereof,
wherein Y represents a C0-C6 linear or branched alkylene group, preferably a C0-C4 linear alkylene group;
r' represents one or more of hydrogen, halogen, C1-C4 alkyl, C1-C4 alkoxy and C1-C4 haloalkyl;
n represents 0, 1, 2 or 3.
5. The unnatural amino acid of any of claims 1-4, wherein the unnatural amino acid is a compound having one of the following structures:
6. use of the unnatural amino acid of any one of claims 1-5 in the preparation of a recombinant protein or recombinant protein conjugate;
preferably, the recombinant protein is recombinant human interleukin 2 or recombinant human growth hormone; and/or
Preferably, the recombinant protein conjugate is a recombinant human interleukin 2-polyethylene glycol conjugate or a recombinant human growth hormone-polyethylene glycol conjugate.
7. A recombinant protein characterized in that at least one site in the amino acid sequence of the recombinant protein is the unnatural amino acid of any one of claims 1-5;
preferably, the recombinant protein is recombinant human interleukin 2 or recombinant human growth hormone.
8. A recombinant protein conjugate, whichCharacterized in that the recombinant protein of claim 7 contains NH and terminal carbonyl groups of unnatural amino acids 2 The coupling moiety of the-O- "end group forms an oxime bond.
9. A recombinant human interleukin 2-polyethylene glycol conjugate comprising recombinant human interleukin 2 comprising at least one unnatural amino acid and PEG conjugated to said at least one unnatural amino acid, wherein said unnatural amino acid is an unnatural amino acid according to any one of claims 1-5, wherein said unnatural amino acid is conjugated to a polypeptide comprising "NH" through its terminal carbonyl group 2 -O- "the PEG of the end group forms an oxime bond to couple the PEG to the at least one unnatural amino acid;
preferably, the recombinant human interleukin 2 is a protein shown as SEQ ID NO. 3 or a functional active fragment thereof; more preferably, the recombinant human interleukin 2 comprising at least one unnatural amino acid is selected from the group consisting of one or more positions corresponding to position P34, position K35, position T37, position R38, position L40, position T41, position F42, position K43, position F44, position Y45, position E61, position E62, position K64, position P65, position E67, position E68, position N71, position L72 and position Y107 of SEQ ID NO. 2;
preferably, said catalyst contains "NH 2 The molecular weight of the PEG of the-O- "end group is 20-50 KD.
10. The recombinant human interleukin 2-polyethylene glycol conjugate of claim 9 in the preparation of a medicament for promoting the immune function of interleukin 2, in the preparation of a medicament for treating cancer, or in the preparation of a medicament for augmenting CD8 + Use of T cells in medicine.
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