Non-natural amino acids, and their uses, recombinant proteins containing them, and recombinant protein conjugates

Abstract

The present invention provides a non-natural amino acid that is a compound having the structure represented by chemical formula (I) or a mirror image isomer thereof. The present invention further provides uses for the non-natural amino acid. Furthermore, the present invention further provides a recombinant protein containing the non-natural amino acid and a protein conjugate prepared from the recombinant protein. The non-natural amino acid provided by the present invention is simple and convenient to prepare, is safe in nature, is not easily inactivated when inserted into a protein, has a high binding rate with the coupling moiety, and the resulting conjugate has higher stability. The non-natural amino acid provided by the present invention is applicable in various fields, particularly in the manufacture of recombinant proteins or recombinant protein conjugates:

Description

Technology Field

[0001] The present invention relates to the field of biopharmaceuticals, and in particular, to non-natural amino acids, recombinant proteins containing such non-natural amino acids, and conjugates formed from such recombinant proteins. Background Technology

[0002] Various scientific research and development and product applications can be realized by introducing non-natural amino acids containing special groups into proteins. For example, the introduction of photosensitive non-natural amino acids into proteins or special labeling of non-natural amino acids is convenient for studying interactions between proteins. Another example is non-natural amino acids used for the directional evolution of enzymes to improve enzyme activity or stability, or to promote efficient enzyme immobilization. As yet another example, safe live bacterial or live viral vaccines can be manufactured by utilizing the characteristic of inserting non-natural amino acids that cannot be realized in normal hosts. A significant application of using codon expansion technology in introducing non-natural amino acids into proteins lies in site-specific modification of proteins, which can enhance protein function, stability, and half-life, and can be used in the development of innovative biological drugs. Currently, a series of achievements have been made, such as the production of sustained-release recombinant proteins from PEG site-specific coupled recombinant human growth hormone and the development of antibody-drug conjugates from small molecule toxin site-specific coupled monoclonal antibodies. Therefore, it can be seen that non-natural amino acids have a very important role and a very wide range of applications.

[0003] Prior art (e.g., CN 102838671B, CN 106146663A, and J. Am. Chem. Soc. 2009, 131, 8720) disclosed Lys-azido, a non-natural amino acid having the following structural formula:

[0004]

[0005] The azide structure (-N3) at the end of Lys-azide is an alkyne-containing structure (e.g., BCN, i.e., A carrier drug (e.g., PEG) modified by ) can be chemically linked to form a conjugate (e.g., Chinese Patent CN 103153927B), which has very high specific selectivity. However, expensive alkyne structures must be introduced in these coupling and chemical modification methods, and an acceptable drug-antibody coupling ratio can only be obtained when alkyne tanks are used in larger equivalents, which increases corresponding production costs and requires complex technical processes and harsh technical conditions.

[0006] Therefore, in order to expand the types and applications of amino acids, it is necessary to develop non-natural amino acids with novel structures using simple manufacturing methods and at low cost.

[0007] In order to overcome the defects in the prior art, one objective of the present invention is to provide non-natural amino acids.

[0008] Another objective of the present invention is to provide the use of non-natural amino acids.

[0009] Another objective of the present invention is to provide a recombinant protein and a recombinant protein conjugate.

[0010] The non-natural amino acid provided by the present invention is a compound having a structure represented by the chemical formula (I) or a mirror image isomer thereof:

[0011] I

[0012] In the above formula,

[0013] X and Z each represent independently substituted or unsubstituted C0-C20 linear or branched alkylenes, one or more -CH2- are optionally substituted by one or more of -O-, -S-, -NH-, -C(O)- and -S(O)-, Y represents -C(O)-, -S(O)- or -CH2-, and A represents a substituted or unsubstituted C6-C20 aryl;

[0014] Where X, Z, and A each independently represent a substituent, such substituent may be selected from one or more of hydroxyl, sulfhydryl, halogen, nitro, cyano, alkyl, alkenyl, alkynyl, alkoxy, acyl, acylamino, carboxyl, ester, amino, sulfonyl, sulfinyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl.

[0015] As shown in Example 11, the inventors of the present invention have discovered that, in addition to high costs and complex processes, when an azide structure (-N3) at the end of Lys-azido is inserted into a protein, such an azide structure is easily reduced to an amino structure (-NH2), thereby losing coupling activity, and that the reduction reaction reduces the yield in the technical manufacturing process.

[0016] .

[0017] A carbonyl is introduced as an active reactive group at the terminal end of the non-natural amino acid provided by the present invention, so that the non-natural amino acid is not only novel in structure and simple and convenient to manufacture, but also mild under coupling conditions, low production costs, and does not easily lose reactive activity due to structural changes when inserted into a protein sequence. The non-natural amino acid provided by the present invention additionally contains an aryl group connected to the terminal carbonyl, and the introduction of the aryl further improves the stability of the obtained conjugate, and such conjugate does not easily decompose even under lower pH conditions. In addition, the non-natural amino acid provided by the present invention additionally contains an alkylene having a specific chain length, which allows the compound to have superior flexibility and form various conjugates better.

[0018] In the non-natural amino acids provided by the present invention, "C0-Cn" comprises C0-C1, C0-C2, ..., C0-Cn, where C0 indicates the absence of a group, and the C atoms at both ends of the group are directly connected to form a bond. For example, the "C0-C6" group indicates the presence of 0 to 6 carbon atoms in this portion, which means that the group is absent, or the group contains 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms. The "C6-C10" group indicates the presence of 6 to 10 carbon atoms in this portion, which means that the group contains 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, or 10 carbon atoms.

[0019] In the non-natural amino acids provided by the present invention, "aryl" represents a carbocyclic aromatic system containing one or two rings, wherein the rings may be connected together in a fusion manner. "Aryl" comprises a monocyclic or bicyclic aryl, e.g., an aromatic group of phenyl, naphthyl, and tetrahydronaphthyl. Preferably, the aryl is a C6-C10 aryl, more preferably, the aryl is phenyl and naphthyl, and most preferably, the aryl is phenyl.

[0020] In some preferred embodiments of the present invention, the substituent may be selected from one or more of hydroxyl, sulfhydryl, halogen, nitro, cyano, C1-C6 alkyl, C1-C6 alkoxy, acyl, acylamino, carboxyl, ester, amino, sulfonyl, sulfinyl, C3-C8 cycloalkyl, C3-C8 heterocyclyl, C6-C20 aryl, and C4-C10 heteroaryl.

[0021] In some preferred embodiments of the present invention, X and Z may each independently represent a C0-C10 linear or branched alkylene, wherein one or more -CH2- may optionally be substituted by one or more of -O-, -S-, and -NH-. In some more preferred embodiments of the present invention, X and Z may each independently represent a C0-C6 linear alkylene, wherein one or more -CH2- may optionally be substituted by one or more of -O-, -S-, and -NH-. In some more preferred embodiments of the present invention, X and Z are not simultaneously C0 alkylenes, meaning that X and Z may not be present simultaneously.

[0022] In some preferred embodiments of the present invention, A may represent a substituted or unsubstituted C6-C10 aryl, and more preferably, A may represent a substituted or unsubstituted phenyl or naphthyl.

[0023] In some preferred embodiments of the present invention, the non-natural amino acid may be a compound having a structure as shown in the chemical formula (I-1):

[0024]

[0025] In the above formula,

[0026] X, Y, and A are each independently as defined in any one of the above technical solutions.

[0027] In some preferred embodiments of the present invention, the non-natural amino acid may be a compound having a structure as shown in the chemical formula (I-2):

[0028]

[0029] In the above formula,

[0030] X is as defined in any one of the above technical solutions;

[0031] R1 and R2 each independently represent hydrogen, hydroxyl, sulfhydryl, halogen, nitro, cyano, C1-C6 alkyl, C1-C6 alkoxy, acyl, acylamino, carboxyl, ester, amino, sulfonyl, sulfinyl, C3-C8 cycloalkyl, C3-C8 heterocyclyl, C6-C20 aryl, or C4-C10 heteroaryl.

[0032] In some preferred embodiments of the present invention, the non-natural amino acid is a compound having a structure as shown in formula (I-3), formula (I-4), formula (I-5) or formula (I-6):

[0033]

[0034] In the above formula,

[0035] X' represents a C0-C6 linear alkylene, more preferably a C0-C4 linear alkylene, and one or more -CH2- are optionally substituted by -O- and / or -NH-;

[0036] R1 and R2 are each independently as defined in any one of the above technical solutions.

[0037] The non-natural amino acids provided by the present invention include optically pure enantiomers and racemics.

[0038] In some more preferred embodiments of the present invention, the non-natural amino acid provided by the present invention is a compound having one of the following structures:

[0039]

[0040] The present invention further provides the use of non-natural amino acids according to any one of the above technical solutions in the manufacture of recombinant proteins or recombinant protein conjugates.

[0041] In the use of the present invention, the recombinant protein may be a recombinant protein obtained by inserting a non-natural amino acid according to any one of the technical solutions into any type of protein common in the art at any site and in any amount. 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 may comprise one or more of, but is not limited to, a polymer (e.g., polyethylene glycol having any molecular weight), a protein, a polypeptide, or a small molecule drug.

[0042] In some preferred embodiments of the present invention, the recombinant protein may be recombinant human growth hormone, and the recombinant protein conjugate may be recombinant human growth hormone-polyethylene glycol conjugate.

[0043] The present invention further provides a recombinant protein in which at least one portion of the amino acid sequence of the recombinant protein is a non-natural amino acid according to any one of the technical solutions.

[0044] Additionally, the recombinant protein may have a structure as shown in chemical formula (II):

[0045]

[0046] In chemical formula (II),

[0047] D represents a residue of a non-natural amino acid according to any one of the above technical solutions from which the aminocarboxylic acid portion has been removed, and P1 and P2 represent the linkage portion of the amino and carboxyl groups of the non-natural amino acid within the amino acid sequence, respectively.

[0048] The recombinant protein provided by the present invention can be manufactured by a method general in the art, comprising realizing the cloning and expression of a recombinant protein containing non-natural amino acids by gene codon expansion technology.

[0049] The recombinant protein provided by the present invention may be a recombinant protein obtained by inserting non-natural amino acids according to any one of the above technical solutions into any type of protein common in the art, such as recombinant human growth hormone, at any site and in any amount.

[0050] The present invention further provides a recombinant protein conjugate formed by forming an oxime bond having a terminal carbonyl of a non-natural amino acid in the recombinant protein according to any one of the technical solutions and a coupling moiety containing an "NH2-O-" terminal group.

[0051] Additionally, the recombinant protein conjugate may have a structure as shown in chemical formula (III):

[0052]

[0053] In chemical formula (III),

[0054] D' represents a residue of a recombinant protein according to any one of the above technical solutions from which the terminal carbonyl portion of a non-natural amino acid has been removed, and D" represents a residue of a coupling moiety from which the "NH2-O-" terminal group has been removed.

[0055] In the recombinant protein conjugate provided by the present invention, the coupling moiety may comprise one or more of a polymer (e.g., polyethylene glycol having any molecular weight), a protein, a polypeptide, or a small molecule drug. Different types of coupling moiety may be coupled separately from the recombinant protein, or different types of coupling moiety may be connected first and then coupled to the recombinant protein.

[0056] In some preferred embodiments of the present invention, the recombinant protein may be recombinant human growth hormone, and the coupling moiety containing an "NH2-O-" terminal group may be polyethylene glycol containing an "NH2-O-" terminal group.

[0057] In some more preferred embodiments of the present invention, polyethylene glycol containing "NH2-O-" terminal groups has the following structural formula:

[0058] ,

[0059] Here, the molecular weight of the polyethylene glycol containing "NH2-O-" end groups may be 10 KD to 100 KD, including, but not limited to, molecular weight values ​​of about 10 KD, 20 KD, 30 KD, 40 KD, 50 KD, 60 KD, 70 KD, 80 KD, 90 KD, and 100 KD, or any combination thereof. Preferably, the molecular weight of the polyethylene glycol containing "NH2-O-" end groups may be 20 KD to 50 KD.

[0060] In some more preferred embodiments of the present invention, the amino acid sequence of the recombinant human growth hormone is as shown in SEQ ID NO: 1, and additionally preferably, the corresponding 107th site within the amino acid sequence of SEQ ID NO: 1 is a non-natural amino acid according to any one of the technical solutions.

[0061] Where the recombinant protein is recombinant human growth hormone, the present invention further provides the use of a recombinant protein conjugate according to any one of the above technical solutions in the manufacture of a drug for treating growth and development disorders caused by endogenous growth hormone secretion insufficiency, growth and development disorders caused by Turner syndrome, or adult growth hormone deficiency.

[0062] The technical solution provided by the present invention has the following advantages.

[0063] (1) A terminal carbonyl and an aryl connected to the terminal carbonyl are introduced into the structure of the non-natural amino acid provided by the present invention, and compared to the current terminal azido non-natural amino acid (e.g., Lys-azido), the non-natural amino acid is simple and convenient to manufacture, has excellent safety, is not easily inactivated when inserted into a protein, has a high binding rate with the coupling moiety, and the obtained conjugate has higher stability.

[0064] (2) As an amino acid derivative, the non-natural amino acid provided by the present invention has the characteristics of an amino acid, so the potential types of amino acids can be expanded, and thus the non-natural amino acid can be applied as an amino acid derivative in various fields, particularly in the manufacture of recombinant proteins or recombinant protein conjugates.

[0065] (3) The non-natural amino acids provided by the present invention can be successfully recognized in prokaryotic and eukaryotic expression systems and inserted into proteins. This allows for the production of proteins containing non-natural amino acids at specific sites, thereby enabling the formation of recombinant proteins with different physical and chemical properties and biochemical activities, and expanding the types of proteins and potential ranges of application. Furthermore, the non-natural amino acids of the present invention have higher expression efficiency in proteins and are more practical.

[0066] (4) Since the recombinant protein produced by the present invention contains the non-natural amino acid of the present invention, the terminal carbonyl active group contained in the non-natural amino acid can easily form a protein conjugate (or conjugate), such as a polyethylene glycol conjugate and a polyethylene glycol-active drug conjugate. These protein conjugates can have many improved biological activities (e.g., anti-tumor activity) due to characteristic modifications.

[0067] (5) The present invention further provides a novel protein conjugate platform, in which various types of proteins and various types of coupling moiety can be combined through novel non-natural amino acids contained in the proteins and connected coupling moiety. Brief explanation of the drawing

[0068] Figure 1 is the profile of the expression plasmid pET21-rhGH107 for recombinant human growth hormone in Example 8. FIG. 2 is an SDS-PAGE electrophoretogram of the fermentation product obtained by adding different types of non-natural amino acids in Example 8, wherein each of the various lanes represents the following: Lane 1, protein molecular weight marker; Lane 2, wild-type recombinant human growth hormone; Lane 3, expression product of recombinant human growth hormone with NBOK added; Lane 4, expression product of recombinant human growth hormone with NPAK added; Lane 5, expression product of recombinant human growth hormone with NBPK added; Lane 6, expression product of recombinant human growth hormone with NBGK added; Lane 7, expression product of recombinant human growth hormone with NPOK added; Lane 8, expression product of recombinant human growth hormone with NPOK-2 added; Lane 9, expression product of recombinant human growth hormone with NBGK-2 added; and Lane 10, expression product of recombinant human growth hormone without any non-natural amino acids added. FIG. 3 is an SDS-PAGE electrophoresis of the coupling product of recombinant human growth hormone containing non-natural amino acids coupled with PEG in Example 8, wherein various lanes each represent the following: Lane 1, molecular weight marker; Lane 2, wild-type recombinant human growth hormone; Lane 3, coupling product of recombinant human growth hormone containing NBOK coupled with PEG; Lane 4, coupling product of recombinant human growth hormone containing NPAK coupled with PEG; Lane 5, coupling product of recombinant human growth hormone containing NBPK coupled with PEG; Lane 6, coupling product of recombinant human growth hormone containing NBGK coupled with PEG; Lane 7, coupling product of recombinant human growth hormone containing NPOK coupled with PEG; and Lane 8, coupling product of recombinant human growth hormone containing NPOK-2 coupled with PEG. FIG. 4 is an SDS-PAGE electrophoresis of the PEG-coupled recombinant human growth hormone purified in Example 8, wherein various lanes each represent the following: lane 1, molecular weight marker; lane 2, standard product of recombinant human growth hormone; lane 3, coupling product of recombinant human growth hormone containing NPOK coupled with PEG; and lane 4, coupling product of recombinant human growth hormone containing NBOK coupled with PEG. FIGS. 5a to 5g are cell activity curves in Example 8, wherein FIG. 5a is the cell activity curve of the rhGH standard product from the National Institutes for Food and Drug Control; FIG. 5b is the cell activity curve of Jintropin®; FIG. 5c is the cell activity curve of Polyethylene Glycol Recombinant Human Somatropin Injection®; FIG. 5d is the cell activity curve of rhGH (NPOK); FIG. 5e is the cell activity curve of PEG-rhGH (NPOK); FIG. 5f is the cell activity curve of rhGH (NBOK); and FIG. 5g is the cell activity curve of PEG-rhGH (NBOK). Figure 6 is a fluorescence microscope image of CHO cells transiently expressing the inserted non-natural amino acid in Example 9. Figure 7 is the profile of the expression plasmid pCDNA3.1-trastuzumab-UAG142 in Example 10. Figures 8a to 8c are HIC-HPLC spectra of trastuzumab containing NBPK coupled with toxin in Example 10, trastuzumab containing NBOK, and trastuzumab containing NPOK-2, respectively. Figure 9 is a graph of the inhibitory effects of trastuzumab containing NBOK and DM1-modified trastuzumab containing NBOK on each BT-474 cell in Example 10. Figures 10a and 10b are the mass spectra of rhGH with a mutation into Lys-azido at the 140th site and rhGH with a mutation into NBOK at the 140th site, respectively, in Example 11. FIGS. 11a and FIGS. 11b are SDS-PAGE electrophoresis of the coupling process of rhGH having a mutation into Lys-azido at the 140th site and rhGH having a mutation into NBOK at the 140th site in Example 11 containing 30KD PEG, respectively, wherein the various lanes in FIG. 11a each represent the following: Lane 1, molecular weight marker; Lane 2, wild-type recombinant human growth hormone; Lane 3, rhGH-Lys-azido-140; Lane 4, product of the coupling reaction over 72 hours when rhGH-Lys-azido-140 : 30K BCN-PEG is 1 : 15 (molar ratio, hereinafter the same); and lane 5, product of the coupling reaction for 72 hours when rhGH-Lys-azido-140 : 30K BCN-PEG is 1 : 25; the various lanes in Fig. 11b each represent the following: lane 1, molecular weight marker; lane 2, rhGH-NBOK-140; lane 3, product of the coupling reaction for 6 hours when rhGH-NBOK-140 : 30K BCN-PEG is 1 : 15; lane 4, product of the coupling reaction for 9 hours when rhGH-NBOK-140 : 30K BCN-PEG is 1 : 15; lane 5, product of the coupling reaction for 12 hours when rhGH-NBOK-140 : 30K BCN-PEG is 1 : 15; Lane 6, product of the coupling reaction for 24 hours when rhGH-NBOK-140 : 30K BCN-PEG is 1 : 15; Lane 7, product of the coupling reaction for 48 hours when rhGH-NBOK-140 : 30K BCN-PEG is 1 : 15. Specific details for implementing the invention

[0069] The technical solution of the present invention will be described in further detail below with reference to specific embodiments.

[0070] Unless otherwise specified, all reagents or raw materials used in the embodiments of the present invention are commercially available.

[0071] Example 1: Preparation of Non-Natural Amino Acid NBOK

[0072] The structural formula of NBOK is as follows:

[0073]

[0074] The reaction process is represented by the following diagram:

[0075]

[0076] The manufacturing process included the following steps.

[0077] a) p methyl acetophenone (4.0 mL, 30.0 mmol), solvent DCE (50.0 mL), NBS (6.41 g, 36.0 mmol), and BPO (0.05 g, 0.3 mmol) were added to a reaction flask. The mixture was refluxed at 80°C for 24 hours, and then the vessel was cooled in ice water to precipitate the solids, which were removed by filtration. The solution was washed three times with saturated Na2CO3 and then extracted three times with DCM. The organic phases were combined, dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the unpurified product 1-1 (5.46 g, yield 85%), which was used directly in the next step without purification.

[0078] b) Product 1-1 (2.73 g, 12.80 mmol), solvent dioxane (40 mL), and water (40 mL) were added to a reaction flask, followed by the addition of calcium carbonate (7.68 g, 76.8 mmol). The mixture was refluxed at 105°C for 24 hours, cooled to room temperature, filtered to remove solids, and then extracted three times with DCM. The organic phases were combined, concentrated under reduced pressure, and purified by column chromatography (eluent: PE:EA = 3:1) to obtain Product 1-2 (1.80 g, yield 94%).

[0079] c) p- Nitrophenyl chloroformate (2.90 g, 14.4 mmol) and solvent DCM (10.0 mL) were added into a 2-neck reaction flask and cooled to 0°C. Product 1-2 (1.80 g, 12.0 mmol) and pyridine (1.2 mL, 14.4 mmol) were added and stirred at room temperature for 18 hours. Subsequently, a saturated aqueous sodium carbonate solution (10 mL) was added to the reaction solution, and it was extracted three times with DCM (50 mL). The organic phases were combined, washed twice with water, dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (eluent: PE:EA = 5:1) to obtain Product 1-3 (3.14 g, yield 83%).

[0080] d) Product 1-3 (1.26 g, 4.0 mmol), Fmoc-Lys-OH hydrochloride (1.40 g, 3.33 mmol), solvent dioxane (15 mL), and water (5 mL) were added into a reaction flask, and then triethylamine (1.2 mL, 8.3 mmol) was added. The mixture was reacted at room temperature for 24 hours, then an appropriate amount of 1 M HCl solution was added, extracted with DCM, and concentrated under reduced pressure to obtain unpurified product 1-4, which was used directly in the next step.

[0081] e) Product 1-4 (1.10 g, 0.19 mmol) was dissolved in DCM (10 mL) in a reaction flask, and diethylamine (5.0 mL) was added and reacted at room temperature for 6 hours to precipitate the product. The product was filtered and then pulped three times with DCM to obtain the target product NBOK (1-5, 817 mg, 2-step yield 63%).

[0082] 1 H-NMR(400 MHz, deuterium) δ 8.04(d, J = 8.4 Hz, 2H), 7.55(d, J= 8.0 Hz, 2H), 5.21(s, 2H), 3.74(t, J = 6.0 Hz, 1H), 3.17(t, J = 6.4 Hz, 2H), 2.70(s, 3H), 1.95 - 1.83(m, 2H), 1.62 - 1.52(m, 2H), 1.47 - 1.35(m, 2H).

[0083] Example 2: Preparation of Non-Natural Amino Acid NPAK

[0084] The structural formula of NPAK is as follows:

[0085]

[0086] The reaction process is represented by the following diagram:

[0087]

[0088] The manufacturing process included the following steps.

[0089] a) p -Chloroacetophenone (1.00 g, 6.47 mmol) was added into the reaction flask, diethyl malonate (6.84 g, 47.70 mmol), KHCO3 (0.97 g, 9.70 mmol), and K2CO3 (1.34 g, 9.70 mmol) were added under a nitrogen atmosphere, and subsequently, Pd(dba)2 (0.019 g, 0.030 mmol) and P( t -Bu)3HBF4 (0.021 g, 0.071 mmol) was added. After addition, nitrogen was used for protection, and the mixture was heated to 160°C and reacted for 40 hours. After the completion of the reaction was detected by TLC, water (30 mL) was added to the reaction solution, and it was extracted three times with EA. The organic phases were combined, washed twice with water, dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at 0°C to 5°C to obtain a colorless, transparent liquid. The liquid was purified by column chromatography (eluent: PE:EA = 10: ) to obtain product 2-1 (0.80 g, yield 60%).

[0090] b) LiOH (0.30 g, 11.64 mmol), water (5.0 mL), ethanol (10 mL), and product 2-1 (0.80 g, 3.88 mmol) were added to a reaction flask and stirred at room temperature for 2 hours. After the completion of the reaction was detected by TLC, 2 M HCl solution was added to the reaction solution to adjust the pH value to 1 to 2, and then extracted three times with EA. The organic phases were combined, washed twice with water, dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain product 2-2 (0.5 g, yield 72%).

[0091] c) Product 2-2 (0.20 g, 1.12 mmol) was added into a reaction flask, and N-hydroxysuccinimide (NHS, 0.19 g, 1.68 mmol), DIPEA (0.07 g, 0.56 mmol), and DCM (2.0 mL) were added sequentially. The mixture was cooled to 0°C to 5°C, and a solution of DCC (0.23 g, 1.12 mmol) and DCM (2.0 mL) were added. The mixture was reacted adiabatically for 2 hours, heated to room temperature, and stirred overnight. After the completion of the reaction was detected by TLC, the reaction solution was filtered, washed with DCM, concentrated under reduced pressure, and purified by column chromatography (eluent: PE:EA = 5:1) to obtain Product 2-3 (0.19 g, yield 62%).

[0092] d) Product 2-3 (0.10 g, 0.36 mmol) was added to a reaction flask, and triethylamine (0.04 g, 0.36 mmol), Fmoc-Lys-OH hydrochloride (0.13 g, 0.36 mmol), dioxane (2.0 mL), and water (2.0 mL) were added sequentially. The mixture was stirred at room temperature and reacted for 18 hours. After the completion of the reaction was detected by TLC, the reaction solution was concentrated under reduced pressure, extracted three times with EA, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: DCM:MeOH = 15:1) to obtain Product 2-4 (0.03 g, yield 61%).

[0093] e) Product 2-4 (0.08 g, 0.15 mmol), DCM (1.0 mL), and piperidine (0.04 g, 0.47 mmol) were added to a reaction flask and stirred at room temperature for 3 hours. After the completion of the reaction was detected by TLC, the reaction solution was concentrated under reduced pressure, pulped with petroleum ether (5 mL) for 1 hour, and filtered. The filter cake was pulped again with petroleum ether (5 mL) for 1 hour and filtered, and then the filter cake was pulped four times repeatedly with ethanol to remove residual piperidine, finally obtaining white-like solid 2-5 (0.02 g, yield 43%).

[0094] 1 H-NMR(400 MHz, deuterium) δ 7.85(d, J = 8.2 Hz, 2H), 7.33(d, J = 8.2 Hz, 2H), 3.94(t, J = 6.3 Hz, 1H), 3.56(s, 2H), 3.12(t, J = 6.8 Hz, 2H), 2.54(s, 3H), 1.80 - 1.70(m, 2H), 1.54 - 1.45(m, 2H), 1.40 - 1.224(m, 2H).

[0095] Example 3: Preparation of Non-Natural Amino Acid NBPK

[0096] The structural formula of NBPK is as follows:

[0097]

[0098] The reaction process is represented by the following diagram:

[0099]

[0100] The manufacturing process included the following steps.

[0101] a) p methylacetophenone (4.0 mL, 30.0 mmol), DCE (50.0 mL), NBS (6.41 g, 36.0 mmol), and BPO (0.05 g, 0.3 mmol) were added to the reaction flask, and the mixture was refluxed at 80°C for 24 hours. After the completion of the reaction was detected by TLC, the vessel was cooled in ice water to precipitate the solids, and the solids were removed by filtration. The solution was washed three times with saturated Na2CO3 and extracted three times with DCM. The organic phases were combined, anhydrous sodium sulfate was added for drying, filtered, and concentrated under reduced pressure to obtain the unpurified product 3-1 (5.46 g, yield 85%), which was used directly in the next step without purification.

[0102] b) NaH (0.58 g, 14.64 mmol, 60%) and dry solvent THF (20 mL) were added into a reaction flask, and ethylene glycol (6.7 mL, 122.0 mmol) was slowly added under cooling in an ice bath. The mixture was stirred at room temperature for 1 hour. Subsequently, product 3-1 (2.60 g, 12.2 mmol) was added, heated, and refluxed at 70°C for 48 hours until the reaction was complete. NaH was quenched by slowly adding saturated NH4Cl under cooling in an ice bath, and then the solution was washed with water and extracted three times with EtOAc. The organic phase was combined, dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (eluent: PE:EA = 2:1) to obtain product 3-2 (1.39 g, yield 59%).

[0103] c) Add product 3-2 (1.39 g, 7.2 mmol) and solvent DCM (10 mL) into the reaction flask, and p - Nitrophenyl chloroformate (1.74 g, 8.64 mmol) and pyridine (0.7 mL, 8.64 mmol) were added under cooling in an ice bath. The mixture was stirred at room temperature for 18 hours. After the completion of the reaction was detected by TLC, water was added to the reaction solution for washing, and it was extracted three times with EtOAc. The organic phases were combined, dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (eluent: PE:EA = 3:1) to obtain product 3-3 (2.27 g, yield 88%).

[0104] d) Product 3-3 (2.27 g, 6.32 mmol), solvent dioxane (16 mL), water (4 mL), and Fmoc-Lys-OH hydrochloride (2.13 g, 5.27 mmol) were added to a reaction flask, and triethylamine (1.85 mL, 13.2 mmol) was added. The mixture was stirred at room temperature for 18 hours until the reaction was complete. An appropriate amount of 1 M HCl was added to the solution to adjust the pH to about 2, and the mixture was extracted with ethyl acetate. The organic phases were combined, dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the unpurified product 3-4, which was used directly in the next step.

[0105] e) The product 3-4 from the previous step was dissolved in DCM (10 mL) in a reaction flask, diethylamine (5 mL) was added, and the reaction was carried out at room temperature for 6 hours. After the completion of the reaction was detected by TLC, the reaction solution was concentrated under reduced pressure and purified by column chromatography (eluent: DCM: MeOH: H2O = 40:10:1) to obtain a white solid product (3-5, 0.95 g, 2-step yield 49%).

[0106] 1 H-NMR(400 MHz, deuterium) δ 8.04(d, J = 8.0 Hz, 2H), 7.56(d, J = 8.0 Hz, 2H), 4.72(s, 2H), 4.25(s, 2H), 3.81(s, 2H), 3.74(t, J = 6.0 Hz, 1H), 3.16 - 3.08(m, 2H), 2.70(s, 3H), 1.97 - 1.79(m, 2H), 1.60 - 1.48(m, 2H), 1.48 - 1.35(m, 2H).

[0107] Example 4: Preparation of Non-Natural Amino Acid NPOK

[0108] The structural formula of NPOK is as follows:

[0109]

[0110] The reaction process is represented by the following diagram:

[0111]

[0112] The manufacturing process included the following steps.

[0113] a) Triphosgene (BTC, 2.18 g, 7.35 mmol) and solvent THF (10.0 mL) were added into a reaction flask, and p-hydroxyacetophenone (2.0 g, 14.7 mmol) and pyridine (1.5 mL, 17.64 mmol) were added under cooling in an ice bath. The mixture was reacted at room temperature for 24 hours. After the completion of the reaction was detected by TLC, an appropriate amount of water was added to the reaction solution, and it was extracted three times with EtOAc. The organic phases were combined, dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the unpurified product 4-1 (1.20 g), which was used directly in the next step.

[0114] b) Boc-lysine (1.1 g, 5.0 mmol), solvent DCM (10.0 mL), product 4-1 (1.20 g), and triethylamine (2 mL, 15 mmol) were added to a reaction flask. The mixture was stirred at room temperature for 24 hours. After the completion of the reaction was detected by TLC, an appropriate amount of 1 M HCl was added to the reaction solution to adjust the pH to a weak acid, and the mixture was extracted three times with DCM. The organic phases were combined, dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (eluent: DCM:MeOH = 5:1) to obtain product 4-2 (1.70 g, yield 84%).

[0115] c) Product 4-2 (1.70 g, 4.2 mmol), solvent DCM (5 mL), and trifluoroacetic acid (5 mL) were added to a reaction flask. The mixture was reacted at room temperature for 1 hour. After the completion of the reaction was detected by TLC, the reaction solution was concentrated directly under reduced pressure and purified by column chromatography (eluent: DCM: MeOH: H2O = 40:10:1) to obtain Product 4-3 (1.19 g, yield 92%).

[0116] 1 H-NMR(400 MHz, deuterium) δ 8.09(d, J = 8.6 Hz, 2H), 7.31(d, J = 8.6 Hz, 2H), 3.78(t, J = 6.0 Hz, 1H), 3.27(t, J = 6.8 Hz, 2H), 2.70(s, 3H), 1.98 - 1.87(m, 2H), 1.72 - 1.60(m, 2H), 1.55 - 1.43(m, 2H).

[0117] Example 5: Preparation of Non-Natural Amino Acid NBGK

[0118] The structural formula of NBGK is as follows:

[0119]

[0120] The reaction process is represented by the following diagram:

[0121]

[0122] The manufacturing process included the following steps.

[0123] a) pmethylacetophenone (8.0 mL, 60.0 mmol), solvent DCE (80.0 mL), NBS (12.82 g, 72.0 mmol), and BPO (145 mg, 0.6 mmol) were added to a reaction flask, and the mixture was refluxed at 90°C for 24 hours. After the completion of the reaction was detected by TLC, the vessel was cooled in ice water to precipitate the solids, and the solids were removed by filtration. The solution was washed three times with saturated Na2CO3 and extracted three times with DCM. The organic phases were combined, dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the unpurified product 5-1 (11.12 g, yield 87%), which was used directly in the next step without purification.

[0124] b) 4 Å MS (14 g) and LiOH (1.45 g, 34.54 mmol) were dissolved in DMF (70 mL) in a reaction flask and stirred at room temperature for 20 minutes, then glycine methyl ester hydrochloride (2.0 g, 15.7 mmol) was added and stirred for 45 minutes, then product 5-1 (4.0 g, 18.8 mmol) was added and stirred at room temperature for 18 hours. After the completion of the reaction was detected by TLC, the solids were removed by filtration, the filter cake was washed with EA, the filtrate was washed twice with water, dried with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the unpurified product 5-2, which was used directly in the next step.

[0125] c) The product 5-2 from the previous step was dissolved in dioxane (20 mL) in a reaction flask, and 1 M NaOH was slowly added dropwise and reacted for 2 hours. Subsequently, the completion of the hydrolysis reaction was monitored by TLC to obtain product 5-3. 20 mL of saturated NaHCO3 was added to product 5-3, Fmoc-OSu dissolved in dioxane (10 mL) was slowly added, and the mixture was stirred overnight at room temperature. After monitoring the completion of the reaction by TLC, the reaction solution was adjusted to a weak acid with 1 M HCl, extracted with EA, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: DCM:MeOH = 10:1) to obtain product 5-4 (3.60 g, yield 89%).

[0126] d) Product 5-3 (3.60 g, 8.0 mmol), NBS (1.10 g, 9.6 mmol), EDCI (1.85 g, 9.6 mmol), and solvent DCM (50 mL) were added to a reaction flask. The mixture was reacted at room temperature for 18 hours. After monitoring the completion of the reaction by TLC, the reaction solution was washed three times with water, dried with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain Product 5-5 (3.20 g, yield 75%).

[0127] e) Product 5-5 (3.20 g, 6.0 mmol), solvents dioxane (40 mL), water (10 mL), and Fmoc-Lys-OH hydrochloride (3.0 g, 7.2 mmol) were added to the reaction flask, followed by the addition of triethylamine (2.0 mL, 15.0 mmol). The mixture was stirred at room temperature for 18 hours until the reaction was complete. An appropriate amount of 1 M HCl was added to the solution to adjust the pH to approximately 2, and the mixture was extracted with EA. The organic phase was combined, dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (eluent: DCM: MeOH: AcOH = 20:1:0.5) to obtain Product 5-5 (3.50 g, yield 75%).

[0128] f) Product 5-5 was dissolved in DCM (20 mL) in a reaction flask, and diethylamine (20 mL) was added and reacted at room temperature for 6 hours. After monitoring the completion of the reaction by TLC, the reaction solution was concentrated under reduced pressure and purified by column chromatography (eluent: DCM: MeOH: H2O = 30:10:1) to obtain the final product 5-7 (0.55 g, yield 37%) of white powder.

[0129] 1 H-NMR(400 MHz, deuterium) δ 7.98(d, J = 8.2 Hz, 2H), 7.50(d, J = 8.2 Hz, 2H), 3.84(s, 2H), 3.71(s, 1H), 3.31(s, 2H), 3.17(t, J = 6.9 Hz, 2H), 2.67(s, 3H), 1.97 - 1.73(m, 2H), 1.58 - 1.45(m, 2H), 1.44 - 1.27(m, 2H).

[0130] Example 6: Preparation of Non-Natural Amino Acid NPOK-2

[0131] The structural formula of NPOK-2 is as follows:

[0132]

[0133] The reaction process is represented by the following diagram:

[0134]

[0135] The manufacturing process included the following steps.

[0136] a) p Acetylphenol (2.05 g, 15.0 mmol) and bromoacetic acid (2.50 g, 18.0 mmol) were added into a reaction flask, followed by the addition of an aqueous solution (6 mL) of NaOH (1.20 g, 30 mmol). The mixture was refluxed at 100°C for 24 hours until the reaction was complete. The reaction solution was cooled to room temperature and adjusted to acidity by adding 1 M hydrochloric acid to precipitate the solid, filtered to obtain a white, unpurified product 6-1 (3.32 g, yield 113%), which was used directly in the next step without purification.

[0137] b) The unpurified product 6-1 (3.32 g, 17.0 mmol) from the previous step was dissolved in DCM (50 mL) in a reaction flask, and NHS (2.35 g, 20.4 mmol) and EDCI (3.90 g, 20.4 mmol) were added. The mixture was stirred at room temperature for 18 hours until the reaction was complete. The reaction solution was extracted with DCM. The organic phases were combined, dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (eluent: DCM: MeOH: AcOH = 20:1:0.5) to obtain product 6-2 (1.67 g, yield 38%).

[0138] c) Product 6-2 (1.67 g, 5.7 mmol), solvents dioxane (20 mL) and water (50 mL), and Fmoc-Lys-OH hydrochloride (1.9 g, 4.8 mmol) were added to the reaction flask, followed by the addition of triethylamine (1.7 mL, 12.0 mmol). The mixture was stirred at room temperature for 18 hours until the reaction was complete. An appropriate amount of 1 M HCl was added to the solution to adjust the pH to approximately 2, and the mixture was extracted with EA. The organic phases were combined, dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The obtained crude product 6-3 was dissolved directly in DCM (10 mL), followed by the addition of diethylamine (5 mL). The mixture was stirred at room temperature for 18 hours until the reaction was complete. The reaction solution was concentrated under reduced pressure and purified by column chromatography (eluent: DCM: MeOH: H2O = 40:10:1) to obtain the final product 6-4 (709 mg, 2-step yield 39%).

[0139] 1 H-NMR(400 MHz, deuterium) δ 7.91(d, J = 8.8 Hz, 2H), 6.98(d, J = 8.8 Hz, 2H), 4.60(s, 2H), 3.59(t, J = 6.4 Hz, 1H), 3.19(t, J = 6.8 Hz, 2H), 2.52(s, 3H), 1.87 - 1.65(m, 2H), 1.56 - 1.40(m, 2H), 1.35 - 1.15(m, 2H).

[0140] Example 7: Preparation of Non-Natural Amino Acid NBGK-2

[0141] The structural formula of NBGK-2 is as follows:

[0142]

[0143] The reaction process is represented by the following diagram:

[0144]

[0145] The manufacturing process included the following steps.

[0146] a) An aqueous solution (10 mL) of bromoacetic acid (2.10 g, 15.0 mmol) and NaOH (0.80 g, 20 mmol) was added to a reaction flask and stirred for 10 minutes, and then p-acetylaniline (1.40 g, 10.0 mmol) was added. The mixture was refluxed at 100°C for 18 hours until the reaction was complete. The reaction solution was cooled to room temperature, filtered, and washed with water to obtain a white, unpurified product 7-1 (1.30 g, yield 67%), which was used directly in the next step without purification.

[0147] b) An aqueous solution (20 mL) of product 7-1 (1.30 g, 6.7 mmol) and NaHCO3 (1.70 g, 20.1 mmol) was added to a reaction flask, followed by the addition of Fmoc-OSu (2.80 g, 8.1 mmol) and DMF (20 mL). The mixture was stirred at 60°C for 18 hours until the reaction was complete. The reaction solution was cooled to room temperature and extracted with EA. The aqueous phase was maintained and adjusted to a pH of about 2 with 1 M hydrochloric acid. Subsequently, the reaction solution was extracted with EA to obtain the organic phase, which was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain product 7-2, which was used directly in the next step without purification.

[0148] c) The product 7-2 (about 6.7 mmol) from the previous step, NHS (0.90 g, 8.0 mmol), and EDCI (1.50 g, 8.0 mmol) were dissolved in DMF (50 mL) in a reaction flask. The reaction mixture was stirred at room temperature for 24 hours until the reaction was complete. Water was added to the reaction solution and extracted with DCM to obtain the organic phase, which was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain product 7-3, which was used directly in the next step without purification.

[0149] d) The product 7-3 (about 6.7 mmol) from the previous step, Fmoc-Lys-OH hydrochloride (2.30 g, 5.6 mmol), and triethylamine (2.0 mL, 14.0 mmol) were added to a reaction flask. The reaction mixture was stirred at room temperature for 3 hours until the reaction was complete. Subsequently, the reaction solution was adjusted to a pH of about 2 with 1 M hydrochloric acid, extracted with EA, dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain product 7-4, which was used directly in the next step without purification.

[0150] e) The product 7-4 from the previous step, solvent DCM (20 mL), and diethylamine (10 mL) were added to a reaction flask. The reaction mixture was stirred at room temperature for 12 hours until the reaction was complete. The reaction solution was first concentrated under reduced pressure, acetonitrile (50 mL) was added to dissolve it again, and then concentrated under reduced pressure; this process was repeated three times to remove excess diethylamine. The reaction solution was pulped twice with the addition of DCM to obtain the final product 7-5 (1.65 g, total yield 51%).

[0151] 1 H-NMR(400 MHz, deuterium) δ 7.71(d, J = 8.8 Hz, 2H), 6.51(d, J= 8.8 Hz, 2H), 3.80(s, 2H), 3.53(t, J = 6.8 Hz, 1H), 3.09(t, J = 6.8 Hz, 2H), 2.39(s, 3H), 1.73 - 1.60(m, 2H), 1.42 - 1.33(m, 2H), 1.25 - 1.14(m, 2H).

[0152] Example 8

[0153] Recombinant human growth hormone was prepared in a prokaryotic expression system containing NPOK, NPAK, NBOK, NBPK, NBGK, NPOK-2, and NBGK-2 prepared in Examples 1 to 7, and a PEG site-specific conjugate was prepared.

[0154] (1) Acquisition of Helper Plasmid

[0155] The helper plasmid pSupAR-MbPylRS was purchased from the plasmid preservation organization Addgene (catalog number: #91705). The plasmid is E. coli ( Escherichia coli It can express non-natural amino acids derived from pyrrolicine and tRNA that specifically recognizes tRNA synthase. The helper plasmid was extracted after shaking-flask incubation in LB medium supplemented with 37.5 mg / L chloramphenicol.

[0156] (2) Composition of a recombinant human growth hormone expression plasmid containing a termination codon within the reading frame

[0157] The mRNA sequence of the coding gene for Homo sapiens growth hormone (having the amino acid sequence as shown in SEQ ID NO: 1) was obtained from the database of the National Center for Biotechnology Information of the United States, and a purification tag consisting of six histidines was added to the C-terminus of its translated protein. Meanwhile, the amino acid codon at the 107th position of SEQ ID NO: 1 was changed to an amber codon (TAG), and subsequently, the complete DNA sequence was synthesized by whole-length gene synthesis to obtain the gene sequence of recombinant human growth hormone (SEQ ID NO: 2).

[0158] The amino acid sequence of Homo sapiens growth hormone (SEQ ID NO: 1) is as follows:

[0159] FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF

[0160] The gene sequence of recombinant human growth hormone (SEQ ID NO: 2) is as follows:

[0161] ATGTTTCCTACTATACCACTATCTCGTCTATTCGATAACGCTATGCTTCGGGCCCATCGTCTTCATCAGCTGGCCTTTGACACCTACCAGGAGTTTGAAGAAGCCTATATCCCAAAGGAACAGAAGTATTCATTCCTGCAGAACCCCCAGACCTCCCTCTGTTTCTCAGAGTCTATTCCGACACCCTCCAACAGGGAGGAAACACAACAGAAATCCAACCTAGAGCTGCTCCGCATCTCCCTGCTGCTCATCCAGTCGTGGCTGGAGCCCGTGCAGTTCCTCAGGAGTGTCTTCGCCAACAGCCTGGTGTACGGCGCCTCTTAGAGCAACGTCTATGACCTCCTAAAGGACCTAGAGGAAGGCATCCAAACGCTGATGGGGAGGCTGGAAGATGGCAGCCCCCGGACTGGGCAGATCTTCAAGCAGACCTACAGCAAGTTCGACACAAACTCACACAACGATGACGCACTACTCAAGAACTACGGGCTGCTCTACTGCTTCAGGAAGGACATGGACAAGGTCGAGACATTCCTGCGCATCGTGCAGTGCCGCTCTGTGGAGGGCAGCTGTGGCTTCCTCGAGCACCACCACCACCACCACTAA

[0162] The gene sequence of recombinant human growth hormone (SEQ ID NO: 2) was cloned via one-step subcloning between the two enzyme cleavage sites Nde I and Xho I of pET21a (Novagen, catalog number: #69740-3) to obtain the expression plasmid pET21-rhGH107, which was confirmed to match the sequence predicted by sequencing. pET21-rhGH107 can be used to express recombinant human growth hormone, in which the amino acid codon at the 107th site is changed to an amber codon and the C-terminus of the protein contains a purification tag consisting of six histidines. The profile of the recombinant human growth hormone expression plasmid pET21-rhGH107 is shown in Fig. 1.

[0163] (3) Acquisition of target expression cell lines

[0164] The above helper plasmid pSupAR-MbPylRS and expression plasmid pET21-rhGH107 were co-transformed into competent cells of Escherichia coli OrigamiB(DE3) (Novagen, catalog number: #70911-3), and dual-resistant cell lines were obtained by screening with LB medium containing 100 mg / L ampicillin and 37.5 mg / L chloramphenicol, i.e., recombinant human growth hormone expression cell lines.

[0165] (4) Expression of recombinant proteins with inserted non-natural amino acids

[0166] Screened expression cell lines of recombinant human growth hormone were inoculated into eight parts of 2×YT medium (containing 100 mg / L ampicillin and 37.5 mg / L chloramphenicol, 16 g / L yeast extract, 10 g / L tryptone and 5 g / L NaCl) and cultured at 37°C until the OD600 of the bacterial liquid was 2.0 ± 0.2. IPTG (final concentration of 1 mM) and arabinose (final concentration of 0.2%) were added to each of the eight fractions, NBOK (final concentration of 1 mM) was added to the first fraction, NPAK (final concentration of 1 mM) was added to the second fraction, NBPK (final concentration of 1 mM) was added to the third fraction, NBGK (final concentration of 1 mM) was added to the fourth fraction, NPOK (final concentration of 1 mM) was added to the fifth fraction, NPOK-2 (final concentration of 1 mM) was added to the sixth fraction, NBGK-2 (final concentration of 1 mM) was added to the seventh fraction, and a non-natural amino acid was added to the eighth fraction as a control. After inducing expression by incubating eight fractions at 37°C for 5 to 6 hours, 1 ml of each culture solution was taken, centrifuged at 10,000 rpm for 1 minute, resuspended in PBS until the OD600 reached 10 days, and each fraction was collected for SDS-PAGE electrophoresis. The SDS-PAGE electrophoresis of each cell line is shown in Figure 2. The results in Figure 2 indicated that the expression cell lines to which seven non-natural amino acids were added could express the target protein.

[0167] (5) Purification of recombinant proteins with inserted non-natural amino acids

[0168] Each fraction after the above expression was induced was centrifuged at 10,000 rpm for 5 minutes, the precipitate was collected, 1 / 20 cell growth volume of NTA buffer (20 mM Tris-HCl, 0.5 M NaCl, and 10% glycerol at pH 7.9) and PMSF at a final concentration of 1 mM were added, the cells were homogenized, and sonicated. The solution was centrifuged at 10,000 rpm for 20 minutes, the supernatant was collected, and the His-tagged target protein was adsorbed onto a Ni-NTA chromatography column, followed by elution with NTA buffer (20 mM Tris-HCl, 0.5 M NaCl, 10% glycerol, and 250 mM imidazole at pH 7.9) to obtain a target protein sample of approximately 90% purity (i.e., a recombinant protein with inserted non-natural amino acids). Each sample was placed in a 10K nitrocellulose dialysis bag and dialyzed overnight with pH 7.0 PBS buffer (200 ml of dialysis solution per 1 ml of protein solution), and the dialysis solution was changed once. The protein solution was collected after dialysis, and the protein concentration was detected by SDS-PAGE.

[0169] (6) Coupling reaction between recombinant protein with inserted non-natural amino acids and PEG

[0170]

[0171] As shown in the synthesis pathway above (wherein the direction from R1 to R2 was the direction from the N-terminus to the C-terminus of the amino acid sequence).

[0172] A coupling reaction was carried out as follows, exemplified by the oxidation reaction of 30KD Aminoxy PEG coupled to a recombinant protein with non-natural amino acids inserted: Before the coupling reaction, the obtained target protein was adjusted to 0.5 mg / ml with pH 7.0 PBS buffer, and 30KD Aminoxy PEG solid (from Beijing Jenkem Technology Co., Ltd.) was added at a ratio of 1:15 (molar ratio of recombinant protein to Aminoxy PEG), and the mixture was shaken thoroughly to obtain a clear and transparent solution. Subsequently, the reaction solution was sealed and shaken in a constant temperature shaker (25°C, 100 rpm). The coupling status was analyzed by SDS-PAGE after 48 hours, as shown in Fig. 3. The results of Figure 3 showed that all six target proteins achieved 30 KD PEG coupling, and also that the seven non-natural amino acids were inserted into the target proteins. The coupling product in which PEG is coupled to the recombinant protein with the inserted non-natural amino acids was abbreviated as "PEG-rhGH".

[0173] (7) Purification of PEG-rhGH

[0174] PEG-rhGH after coupling was separated and purified by a butyl HP hydrophobic chromatography column (loading buffer: 50 mM NaH2PO4˙2H2O at pH 8.5 and 0.8 M (NH4)2SO4; elution buffer: 20 mM Tris-HCl at pH 8.5 and 40-fold column volume gradient elution) to obtain pure PEG-rhGH of electrophoretic grade (>95%). The electrophoretic profiles, with PEG-rhGH containing NPOK and PEG-rhGH containing NBOK as examples, are shown in Figure 4.

[0175] Other PEG-rhGH containing non-natural amino acids of the present invention also reached an electrophoretic purity grade after purification.

[0176] (8) Analysis of PEG-rhGH activity

[0177] The specific process is as follows: HEK293-GHR cells (cell line referenced in Chinese Patent No. 2020112649827) were cultured in sufficient quantities at 37°C in 5% CO2 with DMEM medium (Gibco, catalog number: 11995040) containing 0.1 mg / mL hygromycin B (Sangon Biotech (Shanghai) Co., Ltd., catalog number: A600230-0001), 0.75 mg / mL G418 (Sangon Biotech (Shanghai) Co., Ltd., catalog number: A600958-0005), and 10% fetal bovine serum (Gibco, catalog number: 10099141), and 9×10 4 HEK293-GHR cells were seeded into individual wells of a Black 96-well cell culture plate (Corning, catalog number: 3904) at a capacity of 90 μL / well and starved to 16 hours at 37°C in 5% CO2. rhGH standard products (purchased from the National Institutes for Food and Drug Control, referred to as "NIFDC"), and samples of rhGH containing NPOK (i.e., rhGH(NPOK)), PEG-rhGH of PEG-modified NPOK (i.e., PEG-rhGH(NPOK)), PEG-rhGH containing NBOK (i.e., rhGH(NBOK)), PEG-rhGH of PEG-modified NBOK (i.e., rhGH(NBOK)), PEG-rhGH of PEG-modified NBOK (i.e., PEG-rhGH(NBOK)), and the commercially available rhGH drug Zyntropin ® (rhGH drug Jintropin ® )(Changchun GeneScience Pharmaceutical Co., Ltd.) and commercially available PEG-rhGH drug polyethylene glycol recombinant human somatripine injection ®(PEG-rhGH drug Polyethylene Glycol Recombinant Human Somatropin Injection ® )(Changchun GeneScience Pharmaceutical Co., Ltd.) was gradient-diluted to a total of nine concentrations and incubated at 37°C in 5% CO2 for 4 hours. 50 μL of ONE-Glo™ (Promega, catalog number: E6120) detection reagent was added to each well, and the chemiluminescence values ​​were read using a microplate reader. Curves were fitted based on the sample concentrations and microplate reader readings, and the EC50 was calculated. The results are shown in Figures 5A through 5G and Table 1. The results indicated that the cellular activity at each site before coupling was identical to that of the rhGH standard product from NIFDC. After coupling, cellular activity decreased to approximately 25% to 50% of that of the standard product, and the rule of change in biological activity was consistent with that of commercially available PEG-rhGH.

[0178] The cellular activity of the other PEG-rhGH containing the non-natural amino acid of the present invention after coupling was also reduced to 25% to 50% of the standard product, and the rule of change in biological activity was consistent with that of PEG-rhGH sold commercially.

[0179] Table 1

[0180]

[0181] Example 9

[0182] Recombinant GFP protein was prepared in a eukaryotic expression system with the non-natural amino acids NPOK, NPAK, NBOK, NBPK, NBGK, NPOK-2, and NBGK-2 prepared in Examples 1 to 7.

[0183] (1) Acquisition of Helper Plasmid

[0184] The helper plasmid pCMV-MbPylRS was purchased from the plasmid preservation organization EdGene (catalog number: #91706), and the plasmid encoded an aminoacyl tRNA synthase that specifically recognizes a non-natural amino acid derived from pyrrolicin of mammalian cells and the corresponding tRNA (which recognizes the amber codon UAG).

[0185] (2) Construction of an expression vector of a green fluorescent protein containing an amber codon in a gene reading frame

[0186] An expression vector expressing a wild-type green fluorescent protein containing a purification tag (containing the gene coding sequence shown in SEQ ID NO: 3) was point-mutated to obtain an expression plasmid pEGFP40 in which the codon of the amino acid at the 40th position of the reading frame was mutated to an amber codon, and the complete sequence of the expression plasmid pEGFP40 is shown in SEQ ID NO: 4.

[0187] The gene sequence of the wild-type green fluorescent protein containing the purification tag (SEQ ID NO: 3) is as follows:

[0188] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTAGGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGAAGTACGAAGATTACAAGGATGACGACGATAAGTAA

[0189] 발현 플라스미드 pEGFP40의 유전자 서열(SEQ ID NO: 4)은 이하와 같다:

[0190]

[0191] (3) Expression of recombinant GFP protein with inserted non-natural amino acids

[0192] Chinese hamster ovary cells CHO-K1 (catalog number: #CCL-61-ATC) were purchased from the American Type Culture Collection (ATCC) and cultured by attachment with RPMI1640 medium containing 10% fetal bovine serum. In the above step, the helper plasmid pCMV-MbPylRS and the green fluorescent protein expression plasmid pEGFP40 were extracted and transiently transfected with lip2000 transfection reagent (Invitrogen company, catalog number: #12566014) according to the instructions (cells were seeded into 8 wells at 50,000 cells / well in a 24-well plate, and transfected with 500 ng of plasmid per well 24 hours after seeding). During transfection, NPOK (final concentration of 1 mM) was added to the first well, NPAK (final concentration of 1 mM) to the second well, NBOK (final concentration of 1 mM) to the third well, NBPK (final concentration of 1 mM) to the fourth well, NBGK (final concentration of 1 mM) to the fifth well, NPOK-2 (final concentration of 1 mM) to the sixth well, NBGK-2 (final concentration of 1 mM) to the seventh well, and no natural amino acids were added to the eighth well as a control. After incubating the cells in a CO2 incubator for 48 hours, they were observed under a fluorescence microscope, and the results showed distinct green fluorescence from the first well to the seventh well (see Fig. 6). This demonstrates that natural amino acids can be inserted into the green fluorescent protein, allowing for the production of a complete green fluorescent protein without affecting the function of the fluorescent protein.

[0193] Example 10

[0194] Anti-HER2 monoclonal antibodies containing non-natural amino acids were prepared in a eukaryotic expression system with NPOK, NPAK, NBOK, NBPK, NBGK, NPOK-2, and NBGK-2 prepared in Examples 1 to 7, and conjugates containing toxins were prepared.

[0195] (1) Acquisition of Helper Plasmid

[0196] The helper plasmid pCMV-MbPylRS was purchased from the plasmid preservation organization EdGene (catalog number: #91706), and the plasmid encoded an aminoacyl tRNA synthase that specifically recognizes a non-natural amino acid derived from pyrrolicin of mammalian cells and the corresponding tRNA (which recognizes the amber codon UAG).

[0197] (2) Composition of an expression vector of an anti-HER2 antibody (trastuzumab) containing an amber codon within the reading frame

[0198] Heavy and light chain DNA encoding trastuzumab (corresponding amino acid sequences were SEQ ID NO: 5 and SEQ ID NO: 6, respectively) were synthesized by whole gene synthesis and subcloned into the eukaryotic expression vector pCDNA3.1+. Subsequently, the obtained expression vector was point-mutated to obtain the expression plasmid pCDNA3.1-trastuzumab-UAG142, in which the codon of the amino acid at the 142nd position of the heavy chain reading frame was mutated into an amber codon. The profile of the plasmid is shown in Fig. 7, and the complete sequence of the plasmid is shown in SEQ ID NO: 7.

[0199] The heavy chain amino acid sequence of trastuzumab (SEQ ID NO: 5) is as follows:

[0200] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0201] The light chain amino acid sequence of trastuzumab (SEQ ID NO: 6) is as follows:

[0202] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0203] The gene sequence of the expression plasmid pCDNA3.1-trastuzumab-UAG142 (SEQ ID NO: 7) is as follows:

[0204]

[0205] (3) Insertion of non-natural amino acids

[0206] HEK293 cells adopted in suspension were placed in Wayne293™ medium (Quacell Biotechnology, catalog number: A21501) at a volume of 0.3×10 5 Cells were inoculated at a density of 1 / mL and cultured by shaking in a 1 L shake flask containing 240 mL of medium under conditions of 120 rpm, 5% CO2, and 80% humidity. The cell density was approximately 1 × 10⁶. 6 When the volume was reached / mL, the cells were transfected, the helper plasmid pCMV-MbPylRS and the expression plasmid pCDNA3.1-trastuzumab-UAG142 described in steps (1) and (2) were extracted, and the endotoxin was removed for future use. During the transfection of each shaking flask, 120 μg of the expression plasmid and 120 μg of the helper plasmid were used, added into a centrifuge tube and diluted to 7.2 mL with 1×PBS buffer, and then 720 μg of PEI transfection reagent (polyethyleneimine) was added into another centrifuge tube and diluted to 7.2 mL with 1×PBS buffer. After uniformly mixing the two centrifuge tubes, each was left standing for 5 minutes, then the liquids contained in the two centrifuge tubes were slowly and uniformly mixed and left to stand for 10 minutes, and the mixture was slowly added dropwise to 240 mL of cell suspension in a 1 L shaking flask up to a total of 14.4 mL, and the mixture was shaken continuously and slowly during the addition, and the mixture was cultured continuously for 5 days under conditions of 120 rpm, 5% CO2, and 80% humidity, and the cell culture supernatant was collected for antibody purification.

[0207] (4) Refining

[0208] The cell culture supernatant was purified with a 1 ml HiTrap protein A pre-packed column and eluted with an elution buffer of 100 mmol / L glycine and 200 mmol / L acetate at pH 3.5 to obtain purified trastuzumab with non-natural amino acids inserted.

[0209] (5) Coupling

[0210] The synthesis path is as follows (using NBOK as an example):

[0211]

[0212] A monoclonal antibody-toxin conjugate was obtained by coupling purified trastuzumab containing a toxin with an amino-oxygen terminal group (DM1-PEG-ONH2) and a non-natural amino acid inserted therein (wherein the direction from R1 to R2 was the direction from the N-terminus to the C-terminus of the amino acid sequence) in a site-specific manner via an oximation reaction.

[0213] The manufacturing process of DM1-PEG-ONH2 is as follows:

[0214]

[0215] The specific operation of the coupling process is as follows: purified trastuzumab with inserted non-natural amino acids was mixed and dissolved with DM1-PEG-ONH2 at a molar ratio of 1:15, then the pH value was adjusted to 4.0 with 10 M acetic acid, the mixture was shaken in a shaker (25°C, 200 rpm), and after 48 hours, the reaction status of trastuzumab was sampled and detected by HPLC based on the principle of hydrophobic chromatography (HIC-HPLC). The results indicated that, as shown in Figures 8a to 8c, 93.0% of trastuzumab containing NBPK was modified by the toxin, 93.0% of trastuzumab containing NBOK was modified by the toxin, and 87.4% of trastuzumab containing NPOK-2 was modified by the toxin. Trastuzumab coupling ratio = 100% - percentage of remaining uncoupled material.

[0216] The coupled monoclonal antibody-toxin conjugate was buffered through a 50 kDa ultrafiltration centrifuge tube to remove unreacted toxin raw material, and the buffer was 20 mM histidine buffer (pH 6.5).

[0217] The HIC-HPLC analysis conditions are as follows:

[0218] Mobile phase A (2 M ammonium sulfate, 75 mM K2HPO4, pH 7.2±0.2); and

[0219] Mobile phase B (75 mM K2HPO4, 25% isopropanol, pH 7.2±0.2).

[0220]

[0221] (6) Activity analysis

[0222] The BT-474 cell line (ATCC, catalog number: HTB-20) was used to detect the inhibitory effect of the sample on cell proliferation.

[0223] The specific procedure is as follows: BT-474 cells were cultured at 37°C and 5% CO2 with a sufficient amount of ATCC Hybri-Care medium (ATCC, catalog number: 46-X) ​​containing 10% fetal bovine serum (Gibco, catalog number: 10099141). 1.5 × 10⁴ BT-474 cells were seeded into each well of a 96-well cell culture plate (Corning, catalog number: 3599) (including a vehicle control well), and 80 μL / well of cell-free medium was added to the blank control well. Trastuzumab containing NBOK and DM1-modified trastuzumab containing NBOK were gradientally diluted from 17 μg / mL to 0.13 μg / mL in a total of 8 concentrations, and 2 replicate wells were used for each concentration. The diluted samples were transferred to culture plates inoculated with BT-474 cells at a rate of 10 μL / well, and 10 μL of medium was added to the vehicle control wells and blank control wells, respectively, and cultured under conditions of 37°C and 5% CO2. On day 50, 10 μL of CCK-8 (Beyotime, catalog number: C0043) detection reagent was added to each well, and the plates were incubated for 6 hours under conditions of 37°C and 5% CO2, and the optical density was read by a microplate reader at a wavelength of 450 nm. Growth inhibition of the detected samples was calculated by the following formula: Growth inhibition (%) = (OD Compound - OD Blank Control) / (OD Vehicle Control - OD Blank Control) × 100%. The growth inhibition rates of compounds at different concentrations were calculated in Excel, and then growth inhibition curves were plotted using GraphPad Prism7 software, and the IC50 was calculated. The results are shown in Figure 9. The results indicated that the IC50 of DM1-modified trastuzumab containing NBOK was about 3 times lower than that of trastuzumab containing NBOK, showing a significantly enhanced tumor killing effect.

[0224] (7) Safety test of DM1-modified trastuzumab containing non-natural amino acids

[0225] The coupled monoclonal antibody-toxin conjugate was buffer-exchanged using a 50 kDa ultrafiltration centrifuge (Millipore, catalog number: UFC905024#) to remove unreacted toxin raw material. The exchanged buffer was 20 mM histidine buffer (pH 6.0). The protein solution was divided into three parts and adjusted to pH 4.0, pH 6.0, and pH 8.0, respectively, using 1 M citric acid solution or 1 M Tris solution. Each part was placed in a 25°C water bath, sampled after 24, 48, 72, 96, and 120 hours, respectively, and analyzed by HIC-HPLC (detection conditions were the same as described above). The results are shown in Table 2.

[0226] Table 2: Stability of DM1-modified trastuzumab containing NBOK at 25°C under different pH values

[0227]

[0228] The results of Table 2 indicated that the oxime binding formed in DM1-modified trastuzumab containing non-natural amino acids provided by the present invention remained relatively stable at low pH.

[0229] Example 11: Reduction of the active group of Lys-azido

[0230] (1) Analysis of the molecular weight of the product by high-resolution mass spectrometry

[0231] According to the method of expression of secreted growth hormone in reference (Lihua Song, et al., Journal of Biology, 16(1):6-8, 1999), the OmpA signal peptide coding sequence was added to the N-terminus of rhGH on an expression vector according to the signal peptide sequence provided in the reference, and combined with the helper plasmid pSupAR-MbPylRS of Example 8 to construct an OrigamiB(DE3) expressing cell line of rhGH in which the K140 codon was mutated into the amber codon (TAG). Natural rhGH with a mutation into Lys-azido at the 40th region was expressed by adding Lys-azido during fermentation and was named rhGH-Lys-azido-140. Natural rhGH with a mutation into NBOK at the 140th region was expressed by adding NBOK during fermentation and was named rhGH-NBOK-140. Furthermore, purification was performed by the corresponding purification means described in the aforementioned references. The complete molecular weights of rhGH-Lys-azido-140 and rhGH-NBOK-140 were analyzed by liquid chromatography-mass spectrometry (high-resolution mass spectrometer: XevoG2-XS Q-Tof, Waters Corporation; ultra-high-performance liquid chromatography: UPLC (Acquity UPLC I-Class), Waters Corporation).

[0232] As shown in Fig. 10a, a component with a molecular weight 26 Da less than the theoretical molecular weight (22,265 Da) appeared in the rhGH-Lys-azido-140 sample. It was inferred that this component is a product in which the azide structure (-N3) at the end of Lys-azido is reduced to (-NH2). This indicates that when Lys-azido, a commonly used non-natural amino acid, is inserted into a protein, the azide group is unstable and easily reduced to form a reduced product, thereby losing coupling activity. As shown in Fig. 10b, the rhGH-NBOK-140 sample does not have a high proportion of impurities, which indicates that the recombinant protein product prepared by NBOK is more stable than that prepared by Lys-azido.

[0233] (2) Comparison of coupling efficiencies

[0234]

[0235] As shown in the reaction pathway above, 30 KD BCN-PEG (in-house prepared according to Chinese Patent CN112279906A) was coupled to rhGH-Lys-azido-140 in a site-specific manner via a click reaction (wherein the direction from R1 to R2 was the direction from the N-terminus to the C-terminus of the amino acid sequence). The obtained target protein was adjusted to 0.5 mg / ml in PBS buffer at pH 7.0, and BCN-PEG powder was added to the rhGH-Lys-azido solution at ratios of 1:15 and 1:25 (molar ratio of recombinant protein to 30 KD BCN-PEG), respectively, and dissolved by shaking thoroughly to obtain a clear and transparent solution. Subsequently, the reaction solution was sealed and reacted by shaking in a constant temperature shaker (25°C, 70 rpm). Sampling was performed, and reaction results were detected by SDS-PAGE at regular intervals. The reaction was stopped after 72 hours, and the conversion rate was approximately 50% to 70%. The reaction results are shown in Fig. 11a.

[0236] Referring to Example 8, 30 kD aminoxy PEG was coupled to rhGH-NBOK-140 in a site-specific manner by an oximation reaction. Sampling was performed, and the reaction results were detected by SDS-PAGE at regular intervals. The reaction was stopped after 48 hours, and the conversion rate was approximately 90%. The reaction results are shown in Fig. 11b.

[0237] When combined with the molecular weight detection results from high-resolution mass spectrometry, it can be determined that the reduction of Lys-azido prevents the product from coupling with BCN-PEG, thereby reducing the coupling rate. However, when the recombinant protein containing non-natural amino acids of the present invention is coupled with PEG, the conversion rate is clearly higher compared to that of the recombinant protein containing Lys-azido, and the reaction time is clearly shortened, thereby clearly improving the reaction efficiency.

[0238] Unless otherwise specified, the terms used in this invention have the meanings generally understood by those skilled in the art.

[0239] The described embodiments of the present invention are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. A person skilled in the art may make various other substitutions, modifications, and improvements within the scope of the present invention. Accordingly, the present invention is not limited to the above embodiments but is limited only by the claims.

Claims

Claim 1 Non-natural amino acids that are compounds having the structure represented by chemical formula (I-3), chemical formula (I-4), chemical formula (I-5) or chemical formula (I-6): In the above formula, X' represents a C0-C6 linear alkylene or X' represents a C0-C4 linear alkylene, one -CH2- is optionally substituted by -O- or -NH-; R1 and R2 are each independently hydrogen. Claim 2 In claim 1, a non-natural amino acid is a compound having one of the following structures: Claim 3 A non-natural amino acid according to claim 1 or claim 2, intended for use in the manufacture of a recombinant protein, a recombinant protein conjugate, a recombinant human growth hormone, or a recombinant human growth hormone-polyethylene glycol conjugate. Claim 4 A recombinant protein, wherein at least one site in the amino acid sequence of the recombinant protein is a non-natural amino acid according to claim 1 or claim 2. Claim 5 A recombinant protein conjugate, wherein the recombinant protein conjugate is formed by forming an oxime bond with a coupling moiety containing a terminal carbonyl and an "NH2-O-" terminal group of a non-natural amino acid in the recombinant protein according to claim 4. Claim 6 A recombinant protein conjugate according to claim 5, wherein the recombinant protein is recombinant human growth hormone and the coupling moiety containing an "NH2-O-" terminal group is a polyethylene glycol containing an "NH2-O-" terminal group. Claim 7 A recombinant protein conjugate according to claim 5, intended for use in the manufacture of a drug for treating growth and developmental disorders caused by endogenous growth hormone secretion insufficiency, growth and developmental disorders caused by Turner syndrome, or adult growth hormone deficiency. Claim 8 delete Claim 9 delete Claim 10 delete

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