Unnatural amino acids and uses thereof, recombinant proteins containing same, and recombinant protein conjugates

Abstract

The present invention provides an unnatural amino acid, which is a compound having a structure represented by formula (I) or an enantiomer thereof. Also provided is a use of the unnatural amino acid. Also provided is a recombinant protein containing the unnatural amino acid and a protein conjugate produced from the recombinant protein. The unnatural amino acid is easy to produce, has good safety, is not easily inactivated when inserted into a protein, has a high binding rate with a coupling moiety, and the stability of the resulting conjugate is also higher. The unnatural amino acid can be applied in many fields, especially in the production of recombinant proteins or recombinant protein conjugates. [Case 1] TIFF2024526909000056.tif26170

Description

[Technical Field]

[0001] The present invention relates to the field of biopharmaceuticals, and in particular to unnatural amino acids, recombinant proteins containing said unnatural amino acids, and conjugates of said recombinant proteins. [Background technology]

[0002] The introduction of unnatural amino acids containing special groups into proteins can enable a variety of scientific research and product development applications. For example, the introduction of photosensitive unnatural amino acids into proteins or the addition of special marks to unnatural amino acids can facilitate the study of protein-protein interactions. Furthermore, unnatural amino acids can be used to modify the orientation of enzymes, for example, to increase enzyme activity and stability or facilitate efficient enzyme immobilization. Furthermore, the inability of unnatural amino acids to be inserted into normal hosts can be exploited to produce safe live bacterial or live viral vaccines. One important application of the introduction of unnatural amino acids into proteins using codon extension technology is site-specific modification of proteins to alter their function, stability, half-life, and other characteristics, which can be used in the development of novel biopharmaceuticals. Currently, a series of achievements have been made, such as the production of long-acting recombinant proteins using site-specifically coupled PEG to recombinant human growth hormone and the development of antibody-coupled drugs using site-specifically coupled small molecule toxins to monoclonal antibodies. These findings demonstrate that unnatural amino acids have very important functions and a wide range of applications.

[0003] Previous techniques (e.g., CN102838671B, CN106146663A, J. Am. Chem. Soc. 2009, 131, 8720, etc.) have disclosed the unnatural amino acid Lys-azido, whose structural formula is as follows:

[0004] [ka] The azide structure (-N3) at the end of Lys-azido can be used to form alkyne-containing structures (e.g., BCN, i.e. [ka] (See, for example, Chinese Patent CN103153927B) and can be chemically linked to carrier drugs (e.g., PEG) modified with PEG to form conjugates with high specificity. However, these coupling and chemical modification methods require the introduction of costly alkyne structures, and acceptable drug-antibody coupling rates can only be achieved when a large equivalent amount is used, which increases the corresponding production costs, complicates the production process, and imposes strict production conditions.

[0005] Therefore, in order to expand the variety and uses of amino acids, it is desirable to develop unnatural amino acids that have novel structures, can be produced easily, and are inexpensive. Summary of the Invention

[0006] To overcome the above-mentioned drawbacks of the prior art, one object of the present invention is to provide unnatural amino acids.

[0007] Another object of the present invention is to provide uses of the unnatural amino acids.

[0008] It is yet another object of the present invention to provide recombinant proteins and recombinant protein conjugates.

[0009] The unnatural amino acids provided herein are compounds having a structure represented by formula (I) or an enantiomer thereof: [ka] In the formula, X and Z each independently represent a substituted or unsubstituted C0 to C20 linear or branched alkylene group, in which one or more -CH2- groups can be optionally replaced by one or more selected from -O-, -S-, -NH-, -C(O)-, and -S(O)-; Y represents -C(O)-, -S(O)-, or -CH2-; A represents a substituted or unsubstituted C6 to C20 aryl group; When X, Z, and A each independently represent a substituted group, the substituent is one or more selected from a hydroxyl group, a mercapto group, a halogen, a nitro group, a cyano group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an acyl group, an amido group, a carboxyl group, an ester group, an amino group, a sulfonyl group, a sulfinyl group, a cycloalkyl group, a heterocyclo group, an aryl group, and a heteroaryl group.

[0010] As shown in Example 11, the inventors of the present invention found that in addition to the skyrocketing cost and the complicated production process, the azide structure (-N3) at the end of Lys-azido is easily reduced to an amino structure (-NH2) when inserted into a protein, which makes the coupling activity easily lost, and therefore the yield during the production process is reduced due to this reduction reaction. [ka]

[0011] The unnatural amino acids provided by the present invention have a terminal carbonyl group introduced as an active reactive group, which not only provides a novel structure and is easy to produce, but also allows for mild coupling conditions, reduces production costs, and is less likely to undergo structural changes when inserted into a protein sequence, resulting in less loss of reactivity. The unnatural amino acids provided by the present invention also contain an aryl group bonded to the terminal carbonyl group, which increases the stability of the resulting conjugate and makes the conjugate less susceptible to decomposition even under low pH conditions. Furthermore, the unnatural amino acids provided by the present invention also contain an alkylene group of a predetermined chain length, which improves the flexibility of the compounds and makes it easier to form a variety of conjugates.

[0012] In the unnatural amino acids provided herein, "C0-Cn" includes C0-C1, C0-C2, ...C0-Cn. When it refers to C0, it means that this group is absent, and the C atoms at both ends are directly linked to form a bond. For example, the "C0-C6" group refers to a group containing 0 to 6 carbon atoms in the corresponding moiety, i.e., no group is present, 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 refers to a group containing 6 to 10 carbon atoms in the corresponding moiety, i.e., 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, or 10 carbon atoms.

[0013] In the context of unnatural amino acids of the invention, an "aryl group" refers to a carbocyclic aromatic system containing one or two rings, where such rings may be linked together in a fused fashion. An "aryl group" includes monocyclic or bicyclic aryl groups, such as phenyl, naphthyl, and tetrahydronaphthyl aromatic groups. The aryl group is preferably a C6-C10 aryl group, more preferably a phenyl group or a naphthyl group, and most preferably a phenyl group.

[0014] In some preferred embodiments according to the present invention, the substituent is one or more selected from a hydroxyl group, a mercapto group, a halogen, a nitro group, a cyano group, a C1 to C6 alkyl group, a C1 to C6 alkoxy group, an acyl group, an amido group, a carboxyl group, an ester group, an amino group, a sulfonyl group, a sulfinyl group, a C3 to C8 cycloalkyl group, a C3 to C8 heterocyclo group, a C6 to C20 aryl group, and a C4 to C10 heteroaryl group.

[0015] In some preferred embodiments according to the present invention, X and Z each independently represent a C0-C10 linear or branched alkylene group, in which one or more -CH2- groups may be optionally substituted with one or more selected from -O-, -S-, and -NH-. In some more preferred embodiments according to the present invention, X and Z each independently represent a C0-C6 linear alkylene group, in which one or more -CH2- groups may be optionally substituted with one or more selected from -O-, -S-, and -NH-. In some more preferred embodiments according to the present invention, X and Z are not simultaneously C0 alkylene groups; in other words, it is not permitted for both an X group and a Z group to be absent.

[0016] In some preferred embodiments according to the present invention, A represents a substituted or unsubstituted C6 to C10 aryl group, and more preferably A represents a substituted or unsubstituted phenyl group or naphthyl group.

[0017] In some preferred embodiments according to the present invention, the unnatural amino acid is a compound having a structure represented by formula (I-1): [ka] In the formula, X, Z and A are each independently defined in any one of the above technical solutions.

[0018] In some preferred embodiments according to the present invention, the unnatural amino acid is a compound having a structure represented by formula (I-2): [ka] In the formula, X is defined as in any one of the above technical solutions; R1 and R2 each independently represent hydrogen, a hydroxyl group, a mercapto group, halogen, a nitro group, a cyano group, a C1 to C6 alkyl group, a C1 to C6 alkoxy group, an acyl group, an amido group, a carboxyl group, an ester group, an amino group, a sulfonyl group, a sulfinyl group, a C3 to C8 cycloalkyl group, a C3 to C8 heterocyclo group, a C6 to C20 aryl group, or a C4 to C10 heteroaryl group.

[0019] In some relatively preferred embodiments according to the present invention, the unnatural amino acid is a compound having a structure represented by Formula (I-3), Formula (I-4), Formula (I-5), or Formula (I-6): [ka] [ka] [ka] [ka] In the formula, X' represents a C0-C6 linear alkylene group, more preferably a C0-C4 linear alkylene group, in which one or more -CH2- groups can be optionally substituted with -O- and / or -NH-; The R1 and R2 are each independently defined in any one of the above technical solutions.

[0020] The unnatural amino acids provided herein include optically pure enantiomers and racemates.

[0021] In some more preferred embodiments according to the present invention, the unnatural amino acids provided herein are compounds having a structure represented by any one of the following: [ka]

[0022] The present invention further provides the use of an unnatural amino acid described in any one of the above technical solutions in the production of a recombinant protein or recombinant protein conjugate.

[0023] In the use of the present invention, the recombinant protein is a recombinant protein obtained by inserting any number of unnatural amino acids described in any of the above technical solutions into any type of protein commonly found in the art at any site. The recombinant protein conjugate is a conjugate obtained by coupling any of the obtained recombinant proteins with coupling moieties commonly found in the art, where the coupling moiety includes, but is not limited to, one or more selected from polymers (e.g., polyethylene glycol of any molecular weight), proteins, polypeptides, or small molecule drugs.

[0024] In some preferred embodiments according to the present invention, the recombinant protein is recombinant human growth hormone and the recombinant protein conjugate is a conjugate of recombinant human growth hormone and polyethylene glycol.

[0025] The present invention further provides a recombinant protein in which an unnatural amino acid described in any one of the above technical solutions is arranged at at least one site in the amino acid sequence.

[0026] Furthermore, the recombinant protein has a structure represented by formula (II): [ka] In formula (II), D represents a residue obtained by removing the amino carboxylic acid moiety from an unnatural amino acid described in any one of the above-mentioned technical solutions, and P1 and P2 represent the linking moieties in the amino acid sequence between the amino group and the carboxyl group in the unnatural amino acid, respectively.

[0027] The recombinant proteins provided by the present invention can be produced by production methods commonly found in the art, for example, gene codon extension technology can be used to achieve cloning and expression of recombinant proteins containing unnatural amino acids.

[0028] The recombinant protein provided by the present invention is a recombinant protein obtained by inserting any of the unnatural amino acids described in any of the above technical solutions into any type of protein commonly found in the art at any site and in any number, such as recombinant human growth hormone.

[0029] The present invention further provides a recombinant protein conjugate formed by forming an oxime bond between the carbonyl terminal group of an unnatural amino acid in a recombinant protein described in any one of the above technical solutions and an "NH2-O-" terminal group-containing coupling moiety.

[0030] Furthermore, the recombinant protein conjugate has a structure represented by formula (III): [ka] In formula (III), D' represents a residue obtained by removing the carbonyl terminal group of the unnatural amino acid from a recombinant protein described in any one of the above technical solutions, and D" represents a residue obtained by removing the "NH2-O-" terminal group from the coupling moiety.

[0031] In the recombinant protein conjugates provided herein, the coupling moiety comprises one or more selected from a polymer (e.g., polyethylene glycol of any molecular weight), a protein, a polypeptide, or a small molecule drug. Different types of coupling moieties may be coupled to the recombinant protein independently, or different types of coupling moieties may be linked together and then coupled to the recombinant protein.

[0032] In some preferred embodiments according to the present invention, the recombinant protein is recombinant human growth hormone and the "NH2-O-" terminal group-containing coupling moiety is an "NH2-O-" terminal group-containing polyethylene glycol.

[0033] In some more preferred embodiments according to the present invention, the "NH2-O-" end group containing polyethylene glycol has the following structural formula:

[0034] [ka] Here, the molecular weight of the polyethylene glycol containing an "NH-O-" terminal group is 10 to 100 KD, including, but not limited to, molecular weight values such as about 10 KD, about 20 KD, about 30 KD, about 40 KD, about 50 KD, about 60 KD, about 70 KD, about 80 KD, about 90 KD, about 100 KD, or any combination of molecular weight ranges. Preferably, the molecular weight of the polyethylene glycol containing an "NH-O-" terminal group is 20 to 50 KD.

[0035] In some more preferred embodiments of the present invention, the amino acid sequence of the recombinant human growth hormone is as set forth in SEQ ID NO:1, and more preferably, position 107 of the amino acid sequence of SEQ ID NO:1 is an unnatural amino acid as described in any one of the above technical solutions.

[0036] When the recombinant protein is recombinant human growth hormone, the present invention further provides the use of the recombinant protein conjugate described in any one of the above-mentioned technical solutions in the manufacture of a drug for treating growth and development disorders caused by insufficient secretion of endogenous growth hormone, a drug for treating growth and development disorders caused by Turner syndrome, or a drug for treating adult growth hormone deficiency.

[0037] The technical solution provided in the present invention has the following advantages:

[0038] (1) The unnatural amino acids provided by the present invention have a carbonyl terminal group and an aryl group linked to it in their structure. Compared to current unnatural amino acids with an azido terminal group (e.g., Lys-azido), they are easier to prepare, safer, less likely to be inactivated when inserted into proteins, have a higher conjugation rate with coupling moieties, and the resulting conjugates are more stable.

[0039] (2) As amino acid derivatives, the unnatural amino acids provided by the present invention can themselves possess the properties of amino acids, thereby increasing the potential variety of amino acids and making them applicable to many fields, particularly in the production of recombinant proteins or recombinant protein conjugates.

[0040] (3) The unnatural amino acids provided by the present invention can be successfully identified and inserted into proteins in prokaryotic and eukaryotic expression systems to generate proteins containing unnatural amino acids at specific sites, thereby enabling the formation of recombinant proteins with different physicochemical properties and biochemical activities, thereby broadening the variety and potential uses of proteins. Furthermore, the unnatural amino acids of the present invention can be expressed efficiently in proteins, making them more practical.

[0041] (4) The recombinant proteins provided by the present invention contain unnatural amino acids of the present invention, and the terminal active carbonyl group contained therein allows for the easy formation of protein conjugates (or coupling products), such as polyethylene glycol conjugates and polyethylene glycol-active drug conjugates. These protein conjugates have improved performance and therefore a variety of improved biological activities (e.g., antitumor activity).

[0042] (5) The present invention further provides a novel protein conjugate platform, which enables the conjugation of various proteins and various coupling moieties by using novel unnatural amino acids contained in proteins and coupling moieties linked thereto. [Brief explanation of the drawings]

[0043] [Figure 1] 1 shows the profile of recombinant human growth hormone expression plasmid pET21-rhGH107 in Example 8. [Figure 2] 1 shows an SDS-PAGE electrophoresis diagram of the fermentation products obtained in Example 8 to which different types of unnatural amino acids were added. Each lane in the diagram represents the following: Lane 1: protein molecular weight marker; Lane 2: wild-type recombinant human growth hormone; Lane 3: recombinant human growth hormone expression product administered with NBOK; Lane 4: recombinant human growth hormone expression product administered with NPAK; Lane 5: recombinant human growth hormone expression product administered with NBPK; Lane 6: recombinant human growth hormone expression product administered with NBGK; Lane 7: recombinant human growth hormone expression product administered with NPOK; Lane 8: recombinant human growth hormone expression product administered with NPOK-2; Lane 9: recombinant human growth hormone expression product administered with NBGK-2; Lane 10: recombinant human growth hormone expression product to which no unnatural amino acids were added. [Figure 3]FIG. 1 shows an SDS-PAGE electrophoresis diagram of the coupling product of recombinant human growth hormone containing an unnatural amino acid and PEG in Example 8. The lanes in the diagram 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 and PEG; Lane 4: coupling product of recombinant human growth hormone containing NPAK and PEG; Lane 5: coupling product of recombinant human growth hormone containing NBPK and PEG; Lane 6: coupling product of recombinant human growth hormone containing NBGK and PEG; Lane 7: coupling product of recombinant human growth hormone containing NPOK and PEG; and Lane 8: coupling product of recombinant human growth hormone containing NPOK-2 and PEG. [Figure 4] FIG. 1 shows an SDS-PAGE electrophoresis diagram of purified PEG-coupled recombinant human growth hormone in Example 8. The lanes in the diagram represent the following: Lane 1: molecular weight marker; Lane 2: recombinant human growth hormone standard; Lane 3: coupling product of recombinant human growth hormone containing NPOK and PEG; Lane 4: coupling product of recombinant human growth hormone containing NBOK and PEG. [Figure 5A] 5A shows the cell activity graphs in Example 8, in which FIG. 5A is the cell activity graph of the rhGH standard from China Food and Drug Administration. [Figure 5B] FIG. 5B is a graph showing the cell activity of Jintropin (registered trademark) (generic name: injectable recombinant human growth hormone). [Figure 5C] FIG. 5C is a graph showing the cell activity of Jinsaizeng® (generic name: polyethylene glycol recombinant human growth hormone injection). [Figure 5D] FIG. 5D is a graph of the cellular activity of rhGH(NPOK). [Figure 5E] FIG. 5E is a graph of the cellular activity of PEG-rhGH(NPOK). [Figure 5F] FIG. 5F is a graph of the cellular activity of rhGH(NBOK). [Figure 5G] FIG. 5G is a graph of the cellular activity of PEG-rhGH(NBOK). [Figure 6] 10 shows fluorescence microscopy images showing instantaneous expression of unnatural amino acid insertions in CHO cells in Example 9. [Figure 7] 1 shows the profile of the expression plasmid pCDNA3.1-Trastuzumab-UAG142 in Example 10. [Figure 8A] 1 shows HIC-HPLC spectra of NBPK-containing trastuzumab, NBOK-containing trastuzumab, and NPOK-2-containing trastuzumab after toxins were coupled in Example 10. [Figure 8B] 1 shows HIC-HPLC spectra of NBPK-containing trastuzumab, NBOK-containing trastuzumab, and NPOK-2-containing trastuzumab after toxins were coupled in Example 10. [Figure 8C] 1 shows HIC-HPLC spectra of NBPK-containing trastuzumab, NBOK-containing trastuzumab, and NPOK-2-containing trastuzumab after toxins were coupled in Example 10. [Figure 9] FIG. 10 shows the inhibitory effects on BT-474 cells of NBOK-containing trastuzumab and DM1-modified NBOK-containing trastuzumab in Example 10. [Figure 10A] FIG. 11 shows mass spectra of rhGH in which the 140th position is mutated to Lys-azido and rhGH in which the 140th position is mutated to NBOK in Example 11. [Figure 10B] FIG. 11 shows mass spectra of rhGH in which the 140th position is mutated to Lys-azido and rhGH in which the 140th position is mutated to NBOK in Example 11. [Figure 11A]11A shows SDS-PAGE electrophoresis images of the coupling process of rhGH in which position 140 was mutated to Lys-azido and rhGH in which position 140 was mutated to NBOK with 30KD PEG in Example 11. The lanes in Figure 11A represent the following: Lane 1: molecular weight marker, Lane 2: wild-type recombinant human growth hormone, Lane 3: rhGH-Lys-azido-140, Lane 4: the product obtained by coupling rhGH-Lys-azido-140 with 30KD BCN-PEG at a molar ratio of 1:15 (the same applies below) for 72 hours, and Lane 5: the product obtained by coupling rhGH-Lys-azido-140 with 30KD BCN-PEG at a molar ratio of 1:25 for 72 hours. [Figure 11B] Lanes in Figure 11B represent the following: Lane 1: molecular weight marker, Lane 2: rhGH-NBOK-140, Lane 3: product of coupling reaction of rhGH-NBOK-140 with 30KD BCN-PEG at a 1:15 ratio for 6 hours, Lane 4: product of coupling reaction of rhGH-NBOK-140 with 30KD BCN-PEG at a 1:15 ratio for 9 hours, Lane 5: product of coupling reaction of rhGH-NBOK-140 with 30KD BCN-PEG at a 1:15 ratio for 12 hours, Lane 6: product of coupling reaction of rhGH-NBOK-140 with 30KD BCN-PEG at a 1:15 ratio for 24 hours, Lane 7: product of coupling reaction of rhGH-NBOK-140 with 30KD BCN-PEG at a 1:15 ratio for 48 hours. DETAILED DESCRIPTION OF THE INVENTION

[0044] The technical solution of the present invention will be further described in detail below with reference to specific examples.

[0045] Reagents or materials used in the examples of the present invention are commercially available products unless otherwise specified.

[0046] Example 1: Production of unnatural amino acid NBOK The structural formula of NBOK is shown below.

[0047] [ka] The reaction process was as shown in the diagram below.

[0048] [ka] The manufacturing process includes the following steps:

[0049] a) A reaction flask was charged with p-methylacetophenone (4.0 mL, 30.0 mmol), DCM (50.0 mL), NBS (6.41 g, 36.0 mmol), and BPO (0.05 g, 0.3 mmol). The resulting mixture was refluxed at 80°C for 24 hours, and then the vessel was cooled in ice water to precipitate a solid. The solid was filtered off, washed three times with saturated Na2CO3, and extracted three times with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude product 1-1 (5.46 g, 85% yield). The crude product 1-1 was used directly in the next step without further purification.

[0050] b) Product 1-1 (2.73 g, 12.80 mmol) was added to a reaction flask, and the solvents dioxane (40 mL) and water (40 mL) were added. Calcium carbonate (7.68 g, 76.8 mmol) was then added. The resulting mixture was refluxed at 105°C for 24 hours, then cooled to room temperature, filtered off the solid, extracted three times with DCM, and the organic phases were combined and concentrated under reduced pressure. After purification by column chromatography (eluent: PE:EA = 3:1), product 1-2 (1.80 g, yield 94%) was obtained.

[0051] c) A two-necked reaction flask was charged with p-nitrophenyl chloroformate (2.90 g, 14.4 mmol) and DCM (10.0 mL). The mixture was cooled to 0°C, and product 1-2 (1.80 g, 12.0 mmol) and pyridine (1.2 mL, 14.4 mmol) were added. After stirring at room temperature for 18 hours, saturated sodium carbonate solution (10 mL) was added to the reaction mixture, which was extracted three times with DCM (50 mL). The organic phases were combined, washed twice with water, dried over 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, 83% yield).

[0052] d) Product 1-3 (1.26 g, 4.0 mmol) and Fmoc-Lys-OH hydrochloride (1.40 g, 3.33 mmol) were added to a reaction flask, and the solvent dioxane (15 mL) and water (5 mL) were added, followed by triethylamine (1.2 mL, 8.3 mmol). The resulting mixture was reacted at room temperature for 24 hours, and then an appropriate amount of 1 M HCl solution was added. The mixture was extracted with DCM and concentrated under reduced pressure to obtain crude product 1-4, which was used directly in the next step without further purification.

[0053] e) In a reaction flask, product 1-4 (1.10 g, 0.19 mmol) was dissolved in DCM (10 mL), diethylamine (5.0 mL) was added, and the mixture was allowed to react at room temperature for 6 hours. The product was precipitated, filtered, and then beaten three times with DCM to obtain the desired product NBOK (1-5, 817 mg, 63% yield for two steps).

[0054] 1 H-NMR (400MHz, heavy water) δ8.04(d,J=8.4Hz,2H),7.55(d,J=8.0Hz,2H),5.21(s,2H),3.74(t,J=6.0Hz, 1H),3.17(t,J=6.4Hz,2H),2.70(s,3H),1.95-1.83(m,2H),1.62-1.52(m,2H),1.47-1.35(m,2H).

[0055] Example 2: Preparation of Unnatural Amino Acid NPAK The structural formula of NPAK is shown below.

[0056] [ka] The reaction process was as shown in the diagram below.

[0057] [ka] The manufacturing process includes the following steps:

[0058] a) p-Chloroacetophenone (1.00 g, 6.47 mmol) was added to a reaction flask, and under a nitrogen atmosphere, 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, followed by Pd(dba)2 (0.019 g, 0.030 mmol) and P(t-Bu)3HBF4 (0.021 g, 0.071 mmol). After the addition was complete, the nitrogen protection was replaced, and the temperature was raised to 160°C and the reaction was carried out for 40 hours. When the reaction was complete as determined by TLC, water (30 mL) was added to the reaction mixture, which was then extracted with EA three times. The combined organic phases were washed twice with water, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at 0-5°C to obtain a colorless, transparent liquid. This was purified by column chromatography (eluent: PE:EA = 10:1) to obtain product 2-1 (0.80 g, yield 60%).

[0059] b) A reaction flask was charged with LiOH (0.30 g, 11.64 mmol) and water (5.0 mL), ethanol (10 mL), and product 2-1 (0.80 g, 3.88 mmol). The mixture was stirred at room temperature for 2 hours. When the reaction was complete as determined by TLC, the reaction mixture was adjusted to pH 1-2 by adding 2M HCl solution, extracted three times with EA, and the organic phases were combined, washed twice with water, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain product 2-2 (0.5 g, 72% yield).

[0060] c) Product 2-2 (0.20 g, 1.12 mmol) was added to a reaction flask, followed by N-hydroxysuccinimide (NHS, 0.19 g, 1.68 mmol), DIPEA (0.07 g, 0.56 mmol), and DCM (2.0 mL) in that order. After cooling to 0-5 °C, a solution of DCC (0.23 g, 1.12 mmol) and DCM (2.0 mL) was added and allowed to react for 2 hours. The mixture was then warmed to room temperature and stirred overnight. When the reaction was complete as determined by TLC, the mixture was filtered and washed with DCM. The mother liquor was concentrated under reduced pressure and purified by column chromatography (eluent: PE:EA = 5:1) to obtain product 2-3 (0.19 g, 62% yield).

[0061] d) Product 2-3 (0.10 g, 0.36 mmol) was added to a reaction flask, followed by 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) in that order, and the mixture was stirred at room temperature for 18 hours. When the reaction was complete as determined by TLC, the mixture was concentrated under reduced pressure, extracted three times with EA, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: DCM:MeOH = 15:1) to obtain oily liquid product 2-4 (0.03 g, 61% yield).

[0062] 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. When the reaction was complete as determined by TLC, the mixture was concentrated under reduced pressure, beaten with petroleum ether (5 mL) for 1 hour, filtered, and the resulting filter cake was beaten with petroleum ether (5 mL) for 1 hour, filtered, and the resulting filter cake was beaten with ethanol four times to remove residual piperidine, finally obtaining an off-white solid 2-5 (0.02 g, 43% yield).

[0063] 1H-NMR (400MHz, heavy water) δ7.85(d,J=8.2Hz,2H),7.33(d,J=8.2Hz,2H),3.94(t,J=6.3Hz,1H),3.56(s,2 H),3.12(t,J=6.8Hz,2H),2.54(s,3H),1.80-1.70(m,2H),1.54-1.45(m,2H),1.40-1.224(m,2H).

[0064] Example 3: Production of unnatural amino acid NBPK The structural formula of NBPK is shown below.

[0065] [ka] The reaction process was as shown in the diagram below.

[0066] [ka] The manufacturing process includes the following steps:

[0067] a) A reaction flask was charged with p-methylacetophenone (4.0 mL, 30.0 mmol), DCM (50.0 mL), NBS (6.41 g, 36.0 mmol), and BPO (0.05 g, 0.3 mmol). The resulting mixture was refluxed at 80 °C for 24 h. When the reaction was complete as determined by TLC, the vessel was cooled in ice water to precipitate a solid. The solid was filtered off, washed three times with saturated Na2CO3, and extracted three times with DCM. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude product 3-1 (5.46 g, 85% yield). The crude product 3-1 was used directly in the next step without further purification.

[0068] b) To a reaction flask, NaH (0.58 g, 14.64 mmol, 60%) and dry THF (20 mL) were added. Under ice bath cooling, ethylene glycol (6.7 mL, 122.0 mmol) was slowly added and stirred at room temperature for 1 hour. Then, product 3-1 (2.60 g, 12.2 mmol) was added and heated to reflux at 70 °C for 48 hours to complete the reaction. Under ice bath cooling, saturated NH4Cl was slowly added dropwise to quench the NaH, washed with water, and extracted three times with EtOAc. The organic phases were combined, dried over 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, 59% yield).

[0069] c) Product 3-2 (1.39 g, 7.2 mmol) was added to a reaction flask, and DCM (10 mL) was added. Under ice bath cooling, p-nitrophenyl chloroformate (1.74 g, 8.64 mmol) and pyridine (0.7 mL, 8.64 mmol) were added, and the mixture was stirred at room temperature for 18 hours. When the reaction was complete as determined by TLC, the mixture was washed with water and extracted three times with EtOAc. The organic phases were combined, dried over 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, 88% yield).

[0070] d) Product 3-3 (2.27 g, 6.32 mmol) was added to a reaction flask, followed by the solvents dioxane (16 mL) and water (4 mL). Fmoc-Lys-OH hydrochloride (2.13 g, 5.27 mmol) and triethylamine (1.85 mL, 13.2 mmol) were added, and the mixture was stirred at room temperature for 18 hours to complete the reaction. The pH was adjusted to approximately 2 with 1 M HCl, extracted with ethyl acetate, and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude product 3-4, which was used directly in the next step without further purification.

[0071] e) In a reaction flask, the product 3-4 obtained in the previous step was dissolved in DCM (10 mL), and diethylamine (5 mL) was added and reacted at room temperature for 6 hours. When the reaction was complete as determined by TLC, the mixture was concentrated under reduced pressure and purified by column chromatography (eluent: DCM:MeOH:HO = 40:10:1) to obtain a white solid product (3-5, 0.95 g, 49% yield for two steps).

[0072] 1 H-NMR (400MHz, heavy water) δ8.04(d,J=8.0Hz,2H),7.56(d,J=8.0Hz,2H),4.72(s,2H),4.25(s,2H),3.81(s,2H),3.7 4(t,J=6.0Hz,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).

[0073] Example 4: Production of unnatural amino acid NPOK The structural formula of NPOK is shown below.

[0074] [ka] The reaction process was as shown in the diagram below.

[0075] [ka] The manufacturing process includes the following steps:

[0076] a) Triphosgene (BTC, 2.18 g, 7.35 mmol) was added to a reaction flask, followed by the addition of THF (10.0 mL). Under ice bath cooling, p-hydroxyacetophenone (2.0 g, 14.7 mmol) and pyridine (1.5 mL, 17.64 mmol) were added, and the resulting mixture was reacted at room temperature for 24 hours. When the reaction was complete as determined by TLC, an appropriate amount of water was added, followed by extraction with EtOAc three times, and the organic phases were combined. After drying with anhydrous sodium sulfate, filtration, and concentration under reduced pressure, crude product 4-1 (1.20 g) was obtained, which was used directly in the next step.

[0077] b) Boc-lysine (1.1 g, 5.0 mmol) was added to a reaction flask, followed by DCM (10.0 mL), product 4-1 (1.20 g), and triethylamine (2 mL, 15 mmol). After stirring at room temperature for 24 hours, the reaction was confirmed to be complete by TLC. The pH was adjusted to slightly acidic by adding an appropriate amount of 1 M HCl, and the mixture was extracted three times with DCM. The organic phases were combined. The mixture was dried over 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, 84% yield).

[0078] c) Product 4-2 (1.70 g, 4.2 mmol) was added to a reaction flask, followed by the addition of DCM (5 mL) and trifluoroacetic acid (5 mL). The resulting mixture was allowed to react at room temperature for 1 hour. When the reaction was complete as determined by TLC, the mixture was concentrated under reduced pressure and purified by column chromatography (eluent: DCM:MeOH:HO = 40:10:1) to obtain product 4-3 (1.19 g, 92% yield).

[0079] 1 H-NMR (400MHz, heavy water) δ8.09(d,J=8.6Hz,2H),7.31(d,J=8.6Hz,2H),3.78(t,J=6.0Hz,1H),3. 27(t,J=6.8Hz,2H),2.70(s,3H),1.98-1.87(m,2H),1.72-1.60(m,2H),1.55-1.43(m,2H).

[0080] Example 5: Production of unnatural amino acid NBGK The structural formula of NBGK is shown below.

[0081] [ka] The reaction process was as shown in the diagram below.

[0082] [ka] The manufacturing process includes the following steps:

[0083] a) A reaction flask was charged with p-methylacetophenone (8.0 mL, 60.0 mmol), DCM (80.0 mL), NBS (12.82 g, 72.0 mmol), and BPO (145 mg, 0.6 mmol). The resulting mixture was refluxed at 90 °C for 24 h. When the reaction was complete as determined by TLC, the vessel was cooled in ice water to precipitate a solid. The solid was filtered off, washed three times with saturated Na2CO3, extracted three times with DCM, and the organic phases were combined. After drying with anhydrous sodium sulfate, filtration, and concentration under reduced pressure, crude product 5-1 (11.12 g, 87% yield) was obtained. The crude product 5-1 was used directly in the next step without further purification.

[0084] b) In a reaction flask, 4 Å MS (14 g) and LiOH (1.45 g, 34.54 mmol) were dissolved in DMF (70 mL) and stirred at room temperature for 20 minutes. Glycine methyl ester hydrochloride (2.0 g, 15.7 mmol) was added, and the mixture was stirred for another 45 minutes. Product 5-1 (4.0 g, 18.8 mmol) was added, and the mixture was stirred at room temperature for 18 hours. When the reaction was complete as determined by TLC, the solid was filtered off, and the resulting filter cake was washed with EA. The resulting filtrate was washed twice with water. After drying over anhydrous sodium sulfate and concentration under reduced pressure, crude product 5-2 was obtained and used directly in the next step.

[0085] c) In a reaction flask, the product 5-2 obtained in the previous step was dissolved in dioxane (20 mL), and 1 M NaOH was slowly added dropwise. After reacting for 2 hours, the hydrolysis reaction was completed as determined by TLC to give product 5-3. 20 mL of saturated NaHCO3 was added, followed by the slow addition of Fmoc-OSu dissolved in dioxane (10 mL). The mixture was stirred at room temperature overnight. When the reaction was complete as determined by TLC, the mixture was adjusted to a weak acidity with 1 M HCl, extracted with EA, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: DCM:MeOH = 10:1) to give product 5-4 (3.60 g, 89% yield).

[0086] d) Product 5-3 (3.60 g, 8.0 mmol), NBS (1.10 g, 9.6 mmol), EDCI (1.85 g, 9.6 mmol) were added to a reaction flask, and the solvent DCM (50 mL) was added. The resulting mixture was reacted at room temperature for 18 hours. When the reaction was complete as determined by TLC, the mixture was washed with water three times, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain product 5-5 (3.20 g, 75% yield).

[0087] e) Product 5-5 (3.20 g, 6.0 mmol) was added to a reaction flask, followed by dioxane (40 mL) and water (10 mL). Fmoc-Lys-OH hydrochloride (3.0 g, 7.2 mmol) and triethylamine (2.0 mL, 15.0 mmol) were added, and the mixture was stirred at room temperature for 18 hours to complete the reaction. The pH was adjusted to approximately 2 with 1 M HCl, extracted with EA, and the combined organic phases were dried over 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, 75% yield).

[0088] f) In a reaction flask, product 5-5 was dissolved in DCM (20 mL), and diethylamine (20 mL) was added and reacted at room temperature for 6 hours. After the reaction was complete as determined by TLC, the mixture was concentrated under reduced pressure and purified by column chromatography (eluent: DCM:MeOH:HO = 30:10:1) to obtain the final product 5-7 (0.55 g, 37% yield) as a white powder.

[0089] 1 H-NMR (400MHz, heavy water) δ7.98(d,J=8.2Hz,2H),7.50(d,J=8.2Hz,2H),3.84(s,2H),3.71(s,1H),3.31(s ,2H),3.17(t,J=6.9Hz,2H),2.67(s,3H),1.97-1.73(m,2H),1.58-1.45(m,2H),1.44-1.27(m,2H).

[0090] Example 6: Production of unnatural amino acid NPOK-2 The structural formula of NPOK-2 is shown below.

[0091] [ka] The reaction process was as shown in the diagram below.

[0092] [ka] The manufacturing process includes the following steps:

[0093] a) p-Acetylphenol (2.05 g, 15.0 mmol) and bromoacetic acid (2.50 g, 18.0 mmol) were added to a reaction flask, followed by an aqueous solution (6 mL) of NaOH (1.20 g, 30 mmol). The resulting mixture was refluxed at 100 °C for 24 hours to complete the reaction. The reaction mixture was cooled to room temperature and acidified with 1 M hydrochloric acid to precipitate a solid. After filtration, a white crude product 6-1 (3.32 g, 113% yield) was obtained. The crude product 6-1 was used directly in the next step without further purification.

[0094] b) In a reaction flask, the crude product 6-1 (3.32 g, 17.0 mmol) obtained in the previous step was dissolved in DCM (50 mL), and NHS (2.35 g, 20.4 mmol) and EDCI (3.90 g, 20.4 mmol) were added. The resulting mixture was stirred at room temperature for 18 hours to allow the reaction to complete. After extraction with DCM, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. After further purification by column chromatography (eluent: DCM:MeOH:AcOH = 20:1:0.5), product 6-2 (1.67 g, 38% yield) was obtained.

[0095] c) Product 6-2 (1.67 g, 5.7 mmol) was added to a reaction flask, followed by dioxane (20 mL) and water (50 mL). Fmoc-Lys-OH hydrochloride (1.9 g, 4.8 mmol) and triethylamine (1.7 mL, 12.0 mmol) were added and stirred at room temperature for 18 hours to complete the reaction. The pH was adjusted to approximately 2 with 1 M HCl, extracted with EA, and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting crude product 6-3 was directly dissolved in DCM (10 mL) and diethylamine (5 mL) was added. The resulting mixture was stirred at room temperature for 18 hours to complete the reaction. After concentration under reduced pressure and purification by column chromatography (eluent: DCM:MeOH:HO = 40:10:1), the final product 6-4 (709 mg, 39% yield for two steps) was obtained.

[0096] 1 H-NMR (400MHz, heavy water) δ7.91(d,J=8.8Hz,2H),6.98(d,J=8.8Hz,2H),4.60(s,2H),3.59(t,J=6.4Hz, 1H),3.19(t,J=6.8Hz,2H),2.52(s,3H),1.87-1.65(m,2H),1.56-1.40(m,2H),1.35-1.15(m,2H).

[0097] Example 7: Production of unnatural amino acid NBGK-2 The structural formula of NBGK-2 is shown below.

[0098] [ka] The reaction process was as shown in the diagram below.

[0099] [ka] The manufacturing process includes the following steps:

[0100] a) A solution of bromoacetic acid (2.10 g, 15.0 mmol) and NaOH (0.80 g, 20 mmol) in water (10 mL) was added to a reaction flask and stirred for 10 minutes. p-Acetanilide (1.40 g, 10.0 mmol) was then added, and the resulting mixture was refluxed at 100 °C for 18 hours to complete the reaction. The reaction mixture was cooled to room temperature, filtered, and washed with water to obtain the white crude product 7-1 (1.30 g, 67% yield), which was used directly in the next step without further purification.

[0101] b) A reaction flask was charged with a solution of product 7-1 (1.30 g, 6.7 mmol) and NaHCO3 (1.70 g, 20.1 mmol) in water (20 mL). Fmoc-OSu (2.80 g, 8.1 mmol) and DMF (20 mL) were then added. The resulting mixture was stirred at 60 °C for 18 hours to allow the reaction to complete. The mixture was cooled to room temperature and extracted with EA. The pH of the remaining aqueous phase was adjusted to about 2 with 1 M hydrochloric acid, and then extracted with EA to obtain an organic phase. Anhydrous sodium sulfate was added for drying, filtered, and concentrated under reduced pressure. Product 7-2 was obtained, and the crude product 7-2 was used in the next step without further purification.

[0102] c) In a reaction flask, the product 7-2 (approximately 6.7 mmol) obtained in the previous step, NHS (0.90 g, 8.0 mmol), and EDCI (1.50 g, 8.0 mmol) were dissolved in DMF (50 mL). The reaction mixture was stirred at room temperature for 24 hours to allow for a complete reaction. Water was added, and the mixture was extracted with DCM to obtain the organic phase. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the product 7-3. The crude product 7-3 was used in the next step without further purification.

[0103] d) To a reaction flask, the product 7-3 (approximately 6.7 mmol) obtained in the previous step, Fmoc-Lys-OH hydrochloride (2.30 g, 5.6 mmol), and triethylamine (2.0 mL, 14.0 mmol) were added. The reaction mixture was stirred at room temperature for 3 hours to allow for a complete reaction. The pH was then adjusted to approximately 2 with 1 M hydrochloric acid and extracted with EA. The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain product 7-4, which was used directly in the next step without further purification.

[0104] e) The product 7-4 obtained in the previous step was added to a reaction flask. The solvents DCM (20 mL) and diethylamine (10 mL) were added. The reaction mixture was stirred at room temperature for 12 hours to allow for a complete reaction. The mixture was first concentrated under reduced pressure, redissolved in acetonitrile (50 mL), and then concentrated under reduced pressure three times to remove excess diethylamine. After beating twice with DCM, the final product 7-5 (1.65 g, 51% overall yield) was obtained.

[0105] 1 H-NMR (400MHz, heavy water) δ7.71(d,J=8.8Hz,2H),6.51(d,J=8.8Hz,2H),3.80(s,2H),3.53(t,J=6.8Hz, 1H),3.09(t,J=6.8Hz,2H),2.39(s,3H),1.73-1.60(m,2H),1.42-1.33(m,2H),1.25-1.14(m,2H).

[0106] Example 8 Recombinant human growth hormone was produced in a prokaryotic expression system using NPOK, NPAK, NBOK, NBPK, NBGK, NPOK-2, and NBGK-2 produced in Examples 1 to 7, and a conjugate in which PEG was site-specifically coupled was prepared.

[0107] (1) Acquisition of helper plasmid The helper plasmid pSupAR-MbPylRS was purchased from the plasmid storage organization Addgene (product number #91705). This plasmid can express tRNA and tRNA synthetase that specifically recognizes pyrrolysine-derived unnatural amino acids in E. coli. The helper plasmid was obtained by extraction after shake flask cultivation in LB medium supplemented with 37.5 mg / L chloramphenicol.

[0108] (2) Construction of a recombinant human growth hormone expression plasmid containing a stop codon in the reading frame The mRNA sequence of the gene encoding Homo sapiens growth hormone (the amino acid sequence is as shown in SEQ ID NO:1) was obtained from the database of the National Center for Biotechnology Information. A six-histidine purification tag was then added to the C-terminus of the translated protein, and the codon at position 107 of SEQ ID NO:1 was changed to an amber codon (TAG). The complete DNA sequence was then synthesized by total gene synthesis to obtain the gene sequence of recombinant human growth hormone (SEQ ID NO:2).

[0109] The amino acid sequence of Homo sapiens growth hormone (SEQ ID NO:1) is as follows:

[0110] [Table 1]

[0111] The gene sequence of the recombinant human growth hormone (SEQ ID NO:2) was as follows:

[0112] [Table 2]

[0113] Furthermore, the recombinant human growth hormone gene sequence (SEQ ID NO:2) was subcloned between the NdeI and XhoI enzyme cleavage sites of pET21a (Novagen, product number #69740-3) to obtain the expression plasmid pET21-rhGH107. The sequence was determined to be identical to the predicted sequence. pET21-rhGH107 can express recombinant human growth hormone in which the amino acid codon at position 107 is replaced with an amber codon, and the protein contains a six-histidine purification tag at its C-terminus. The profile of the recombinant human growth hormone expression plasmid pET21-rhGH107 is shown in Figure 1.

[0114] (3) Obtaining the target expression strain The above helper plasmid pSupAR-MbPylRS and the expression plasmid pET21-rhGH107 were co-transformed into competent Escherichia coli OrigamiB(DE3) (Novagen, product number #70911-3), and the double-resistant strain obtained by screening in LB medium containing 100 mg / L ampicillin and 37.5 mg / L chloramphenicol was identified as a recombinant human growth hormone-expressing strain.

[0115] (4) Expression of recombinant proteins containing unnatural amino acids The recombinant human growth hormone-expressing strains obtained by screening were cultured in eight sets of 2xYT medium (16 g / L yeast extract, 10 g / L tryptone, 5 g / L The unnatural amino acid was added to a 100-kJ / mL medium containing 100 mg / L NaCl, 100 mg / L ampicillin, and 37.5 mg / L chloramphenicol, and the mixture was cultured at 37°C until the OD600 of the culture reached 2.0 ± 0.2. Eight sets of cultures were supplemented with IPTG (final concentration: 1 mM) and arabinose (final concentration: 0.2%). NBOK (final concentration: 1 mM) was added to the first set of cultures, NPAK (final concentration: 1 mM) to the second set of cultures, NBPK (final concentration: 1 mM) to the third set of cultures, NBGK (final concentration: 1 mM) to the fourth set of cultures, NPOK (final concentration: 1 mM) to the fifth set of cultures, NPOK-2 (final concentration: 1 mM) to the sixth set of cultures, NBGK-2 (final concentration: 1 mM) to the seventh set of cultures, and the eighth set of cultures was used as a negative control without the addition of an unnatural amino acid. After 5-6 hours of induced expression culture at 37°C, 1 ml of each culture was taken and centrifuged at 10,000 rpm for 1 minute, then resuspended in PBS until the OD600 reached 10. Each bacterial suspension was subjected to SDS-PAGE electrophoresis, and the SDS-PAGE electrophoresis patterns for each strain are shown in Figure 2. As is clear from the results in Figure 2, the expression strains were able to express the target protein when each of the seven unnatural amino acids was added.

[0116] (5) Purification of recombinant proteins containing unnatural amino acids After the induction and expression, each bacterial solution was centrifuged at 10,000 rpm for 5 minutes. The precipitate was collected and 1 / 20 the cell growth volume of NTA buffer (20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 10% glycerol) and a final concentration of 1 mM PMSF were added. The cells were disrupted by ultrasonic homogenization and centrifuged at 10,000 rpm for 20 minutes. The supernatant was then collected and the His-tagged target protein was adsorbed onto a Ni-NTA chromatography column and eluted with NTA buffer (20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 10% glycerol, 250 mM imidazole) to obtain a target protein sample with approximately 90% purity (i.e., recombinant protein containing an unnatural amino acid inserted). Each sample was placed in a 10K cellulose nitrate dialysis bag and dialyzed overnight in PBS buffer at pH 7.0 (corresponding to 200 ml of dialysate per ml of protein solution), with one fluid exchange. The dialyzed protein solution was collected and the protein concentration was measured by SDS-PAGE.

[0117] (6) Coupling reaction of recombinant proteins containing unnatural amino acids with PEG [ka] The synthetic route was as described above (wherein the direction from R1 to R2 is from the N-terminus to the C-terminus of the amino acid sequence).

[0118] For example, 30 kD aminooxy PEG was coupled to recombinant proteins containing unnatural amino acids via oximation. The coupling procedure was as follows: Before the coupling reaction, the target protein obtained as described above was adjusted to 0.5 mg / ml with PBS buffer at pH 7.0. 30 kD aminooxy PEG solid (purchased from Beijing Keykai Technology Co., Ltd.) was added at a molar ratio of 1:15 (recombinant protein to aminooxy PEG). The mixture was thoroughly dissolved by shaking to obtain a clear, transparent solution. The reaction mixture was then sealed and allowed to react with shaking on a thermostatic shaker (25 °C, 100 rpm). After 48 hours, the coupling status was analyzed using SDS-PAGE (see Figure 3). As can be seen from Figure 3, 30 kD PEG was coupled to all six target proteins, and the seven unnatural amino acids were also inserted into the target proteins. The coupling product of the recombinant proteins containing unnatural amino acids and PEG was abbreviated as "PEG-rhGH."

[0119] (7) Purification of PEG-rhGH The coupled PEG-rhGH was purified using a Butyl HP hydrophobic chromatography column (loading buffer: 50 mM NaH2PO4·2H2O, 0.8 M (NH4)2SO4, pH 8.5; elution buffer: 20 mM Tris-HCl, pH 8.5, gradient elution over 40 column volumes). The resulting pure PEG-rhGH reached a purity level of >95% on electrophoresis. The electrophoretic patterns of NPOK- and NBOK-containing PEG-rhGH are shown in Figure 4.

[0120] Other PEG-rhGH containing unnatural amino acids of the invention similarly reached electrophoretically pure levels after purification.

[0121] (8) Activity analysis of PEG-rhGH The specific process was as follows: HEK293-GHR cells (see Chinese Patent No. 2020112649827 for cell line origin) were cultured in DMEM (Gibco, product number 11995040) medium containing 0.1 mg / mL hygromycin B (Sangon Biotech, product number A600230-0001), 0.75 mg / mL G418 (Sangon Biotech, product number A600958-0005), and 10% fetal bovine serum (Gibco, product number 10099141) at 37°C under 5% CO2 conditions until sufficient numbers of HEK 293-GHR cells were grown in a black 96-well cell culture plate (Corning, product number 3904) at 9 × 10 per well. 4 90 μL / well of rhGH was inoculated and cultured at 37°C under 5% CO2 for 16 hours under starvation conditions. Standard rhGH (purchased from the China Food and Drug Administration, abbreviated as "Chinese Academy"), NPOK-containing rhGH (i.e., rhGH(NPOK)), PEG-rhGH in which NPOK was modified with PEG (i.e., PEG-rhGH(NPOK)), NBOK-containing PEG-rhGH (i.e., rhGH(NBOK)), PEG-rhGH in which NBOK was modified with PEG (i.e., PEG-rhGH(NBOK)), the commercially available rhGH drug, Saizeng® (manufactured by Changchun Jinsai Pharmaceutical Co., Ltd.), and the commercially available PEG-rhGH drug, Jinsaizeng® (manufactured by Changchun Jinsai Pharmaceutical Co., Ltd.) were diluted in a gradient to a total of nine concentrations and cultured at 37°C under 5% CO2 for 4 hours. ONE-Glo TM 50 μL of detection reagent (Promega, product number E6120) was added per well, and the chemiluminescence readings were measured using an enzyme labeling device. A curve was fitted based on the sample concentration and the readings from the enzyme labeling device to calculate the EC50. The results are shown in Figures 5A-5G and Table 1. As can be seen from the results, before the coupling reaction, the cellular activity at each site was consistent with that of the rhGH standard prepared by the Chinese Medical Association. After the coupling reaction, the cellular activity decreased to approximately 25%-50% of that of the standard, and the change in biological activity was consistent with that of commercially available PEG-rhGH.

[0122] The other PEG-rhGH containing unnatural amino acids of the present invention also showed reduced cellular activity after coupling to approximately 25% to 50% of that of the standard product, and the change in biological activity was consistent with that of commercially available PEG-rhGH.

[0123] [Table 3]

[0124] Example 9 The unnatural amino acids NPOK, NPAK, NBOK, NBPK, NBGK, NPOK-2, and NBGK-2 produced in Examples 1 to 7 were used to produce recombinant GFP protein in a eukaryotic expression system.

[0125] (1) Acquisition of helper plasmid The helper plasmid pCMV-MbPylRS was purchased from the plasmid storage organization addgene (product number #91706). This plasmid encodes an aminoacyl-tRNA synthetase that specifically recognizes pyrrolysine-derived unnatural amino acids in mammalian cells, and the corresponding tRNA (which recognizes the amber codon UAG).

[0126] (2) Construction of a green fluorescent protein expression vector containing an amber codon in the gene reading frame An expression vector expressing purified tagged wild-type green fluorescent protein (the gene coding sequence of which was as shown in SEQ ID NO:3) was subjected to point mutation to obtain expression plasmid pEGFP40, in which the codon at position 40 in the reading frame was mutated to an amber codon, and the complete sequence of the plasmid was shown in SEQ ID NO:4.

[0127] The gene sequence of the purified tagged wild-type green fluorescent protein (SEQ ID NO:3) was as follows:

[0128] [Table 4]

[0129] The gene sequence (SEQ ID NO:4) of the expression plasmid pEGFP40 was as follows:

[0130] [Table 5(1)] [Table 5(2)] [Table 5(3)]

[0131] (3) Expression of recombinant GFP protein containing unnatural amino acids Chinese hamster ovary cells CHO-K1 (product number CCL-61-ATC) were purchased from the American Type Culture Collection (ATCC) and cultured in RPMI 1640 medium containing 10% fetal bovine serum. The helper plasmid pCMV-MbPylRS and green fluorescent protein expression plasmid pEGFP40 obtained in the above process were extracted and transfected immediately using lipo2000 transfection reagent (Invitrogen, product number 12566014) according to the manufacturer's instructions. The cells were seeded into 24-well plates at a dose of 50,000 cells / well (a total of eight wells). After 24 hours of incubation, the cells were transfected. 500 ng of plasmid per well was added. (The unnatural amino acid was used as a smid.) Simultaneously with transfection, NPOK (final concentration 1 mM) was added to the first well, NPAK (final concentration 1 mM) to the second well, NBOK (final concentration 1 mM) to the third well, NBPK (final concentration 1 mM) to the fourth well, NBGK (final concentration 1 mM) to the fifth well, NPOK-2 (final concentration 1 mM) to the sixth well, NBGK-2 (final concentration 1 mM) to the seventh well, and no unnatural amino acid was added to the eighth well, which served as a negative control. After 48 hours of static culture in a CO2 incubator, cells were observed under a fluorescence microscope. Clear green fluorescence was observed in wells 1 through 7 (see Figure 6). This demonstrates that the unnatural amino acid was inserted into the green fluorescent protein to obtain an intact green fluorescent protein, without affecting the function of the fluorescent protein.

[0132] Example 10 Using NPOK, NPAK, NBOK, NBPK, NBGK, NPOK-2, and NBGK-2 produced in Examples 1 to 7, anti-HER2 monoclonal antibodies containing unnatural amino acids were expressed in a eukaryotic expression system, and conjugates with toxins were prepared.

[0133] (1) Acquisition of helper plasmid The helper plasmid pCMV-MbPylRS was purchased from the plasmid storage organization addgene (product number #91706). This plasmid encodes an aminoacyl-tRNA synthetase that specifically recognizes pyrrolysine-derived unnatural amino acids in mammalian cells, and the corresponding tRNA (which recognizes the amber codon UAG).

[0134] (2) Construction of an anti-HER2 antibody (trastuzumab) expression vector containing an amber codon in the gene reading frame The heavy and light chain DNAs encoding trastuzumab (the corresponding amino acid sequences are SEQ ID NO: 5 and SEQ ID NO: 6, respectively) were synthesized by total gene synthesis and subcloned into the eukaryotic expression vector pCDNA3.1+. The resulting expression vector was subjected to point mutation to obtain the expression plasmid pCDNA3.1-Trastuzumab-UAG142, in which the amino acid codon at position 142 in the heavy chain reading frame was mutated to an amber codon. The profile is shown in Figure 7, and the complete sequence is shown in SEQ ID NO: 7.

[0135] The heavy chain amino acid sequence of trastuzumab (SEQ ID NO:5) was as follows:

[0136] [Table 6]

[0137] The light chain amino acid sequence of trastuzumab (SEQ ID NO:6) was as follows:

[0138] [Table 7]

[0139] The gene sequence (SEQ ID NO: 7) of the expression plasmid pCDNA3.1-Trastuzumab-UAG142 was as follows:

[0140] [Table 8(1)] [Table 8(2)] [Table 8(3)]

[0141] (3) Insertion of unnatural amino acids HEK293 cells adapted to suspension were used, and 0.3 × 10 5 / mL density Wayne293 TM The medium (Quacell Biotechnology, product number A21501) was inoculated and cultured in a 1 L shake flask with a filling volume of 240 mL at 120 rpm under conditions of 5% CO2 and 80% humidity until the cell density reached approximately 1 × 10 6 When the concentration reached 100 / mL, transfection was performed, and the helper plasmid pCMV-MbPylRS and expression plasmid pCDNA3.1-Trastuzumab-UAG142 described in steps (1) and (2) were extracted, endotoxin was removed, and the procedure proceeded to standby mode. For each shake flask, 120 μg of expression plasmid and helper plasmid were used for transfection. The plasmids were added to a centrifuge tube and diluted to 7.2 mL with 1x PBS buffer. 720 μg of PEI transfection reagent (polyethyleneimine) was added to another centrifuge tube and diluted to 7.2 mL with 1x PBS buffer. After homogenization, the two centrifuge tubes were allowed to stand for 5 minutes. The liquids in the two centrifuge tubes were then gently mixed homogenously and allowed to stand for 10 minutes. A total of 14.4 mL of the resulting mixture was slowly added dropwise to 240 mL of cell suspension in a 1 L shake flask, with constant gentle shaking during the addition. After 4 hours of shaking incubation at 120 rpm, 5% CO2, and 80% humidity, an unnatural amino acid was added to a final concentration of 1 mM. After 5 days of shaking incubation at 120 rpm, 5% CO2, and 80% humidity, the cell culture supernatant for antibody purification was obtained.

[0142] (4) Refining The cell culture supernatant was purified using a 1 ml pre-packed column with Hi Trap Protein A. Purified trastuzumab with unnatural amino acids inserted was obtained using an elution buffer of 100 mmol / L glycine, 200 mmol / L acetate, pH 3.5.

[0143] (5) Coupling The synthesis route (using NBOK as an example) was as follows:

[0144] [ka] A monoclonal antibody-toxin conjugate was obtained by site-specific coupling of a toxin containing an aminooxy terminal group (DM1-PEG-ONH2) with purified trastuzumab containing an unnatural amino acid (where the direction from R1 to R2 is from the N-terminus to the C-terminus of the amino acid sequence) via an oximation reaction.

[0145] The manufacturing process of DM1-PEG-ONH2 was as follows.

[0146] [ka] The specific procedure for the coupling process was as follows: purified unnatural amino acid-incorporated trastuzumab was mixed with DM1-PEG-ONH2 at a molar ratio of 1:15 and dissolved in a clear solution. The pH was then adjusted to 4.0 with 10 M acetic acid and the mixture was shaken on a shaking table (25°C, 200 rpm). After 48 hours, a sample was taken and the reaction status of trastuzumab was measured using hydrophobic chromatography-based HPLC (HIC-HPLC). As shown in Figures 8A-8C, 93.0% of the NBPK-containing trastuzumab was modified with toxin, 93.0% of the NBOK-containing trastuzumab was modified with toxin, and 87.4% of the NPOK-2-containing trastuzumab was modified with toxin. The coupling ratio of trastuzumab = 100% - the percentage of excess raw material that did not undergo coupling reaction.

[0147] The monoclonal antibody-toxin conjugate obtained by coupling was passed through a 50 kDa ultrafiltration centrifuge tube to perform a liquid exchange, removing the toxin raw material that did not react, and the exchange buffer was changed to 20 mM histidine buffer (pH 6.5).

[0148] The HIC-HPLC analysis conditions were as follows.

[0149] Mobile phase A (2 M ammonium sulfate, 75 mM K2HPO4, pH 7.2 ± 0.2), Mobile phase B (75 mM K2HPO4, 25% isopropanol, pH 7.2 ± 0.2).

[0150] [Table 9]

[0151] (6) Activity analysis The inhibitory effect of the samples on cell proliferation was measured using the BT-474 cell line (purchased from ATCC, product number HTB-20).

[0152] The specific process was as follows: BT-474 cells were cultured in ATCC Hybri-Care Medium (purchased from ATCC, product number 46-X) containing 10% fetal bovine serum (manufactured by Gibco, product number 10099141) at 37°C under 5% CO2 conditions until a sufficient number of cells were obtained. Then, BT-474 cells were plated in a 96-well cell culture plate (manufactured by Corning, product number 3599) at a density of 1.5 × 10 cells per well. 4 Each well was inoculated with 10 μL of CCK-8 (Hekiunten Co., Ltd., product number C0043) detection reagent. The wells were incubated at 37°C for 6 hours at 37°C with 5% CO2, and 80 μL of medium without cells was added to the blank control wells. NBOK-containing trastuzumab and DM1-modified NBOK-containing trastuzumab were diluted from 17 μg / mL to 0.13 μg / mL, yielding a total of eight concentrations, with two replicate wells per dilution. The diluted samples were transferred to the BT-474 cell-inoculated culture plate at 10 μL per well. 10 μL of medium was added to the solvent control and blank control wells, and the wells were incubated at 37°C with 5% CO2. On day 5, 10 μL of CCK-8 (Hekiunten Co., Ltd., product number C0043) detection reagent was added per well. The color was developed at 37°C with 5% CO2 for 6 hours, and the optical density was read at 450 nm using an enzyme detector. The growth inhibition rate (growth inhibition) of the test sample was calculated using the following formula: Inhibition rate (%) = (OD compound - OD blank control) / (OD solvent control - OD blank control) × 100%. The inhibition rates of different concentrations of the compound were calculated using Excel, and then an inhibition graph was created using GraphPad Prism 7 software to calculate the IC50. The results are shown in Figure 9. As can be seen from the results, the IC50 of DM1-modified NBOK-containing trastuzumab was approximately three-fold lower than that of NBOK-containing trastuzumab, demonstrating a significantly improved tumor killing effect.

[0153] (7) Stability considerations of trastuzumab containing unnatural amino acids modified with DM1 The monoclonal antibody-toxin conjugate obtained by coupling was passed through a 50 kDa ultrafiltration centrifuge tube (Millipore, part number UFC905024#) to perform a liquid exchange and remove unreacted toxin raw materials. The exchange buffer was 20 mM histidine buffer (pH 6.0). The protein solution was divided into three portions, adjusted to pH 4.0, pH 6.0, and pH 8.0 with 1 M citric acid solution or 1 M Tris solution, and placed in a 25°C water bath. Samples were taken at 24, 48, 72, 96, and 120 hours and detected by HIC-HPLC (detection conditions were the same as above). The results are shown in Table 2.

[0154] [Table 10]

[0155] As is clear from the results in Table 2, in the DM1-modified unnatural amino acid-containing trastuzumab provided by the present invention, the formed oxime bond was relatively stable even at low pH.

[0156] Example 11: Lys-azido active group reduction phenomenon (1) Analysis of product molecular weight by high-resolution mass spectrometry Based on the literature's method for expressing secretory growth hormone (Song Lihua et al., Journal of Biology, 16(1):6-8, 1999), the coding sequence for the OmpA signal peptide was added to the N-terminus of rhGH in the expression vector based on the signal peptide sequence provided in the literature. This was then combined with the helper plasmid pSupAR-MbPylRS from Example 8 to construct an OrigamiB(DE3) expression strain for rhGH in which the K140 codon was mutated to an amber codon (TAG). Lys-azido was added during the fermentation process to express native rhGH in which the 140 position was mutated to Lys-azido, designated rhGH-Lys-azido-140. Furthermore, NBOK was added during the fermentation process to express native rhGH in which the 140 position was mutated to NBOK, designated rhGH-NBOK-140. Purification was carried out according to the purification procedures described in the above reference. Complete molecular weight analysis of rhGH-Lys-azido-140 and rhGH-NBOK-140 was performed by liquid chromatography and mass spectrometry (high-resolution mass spectrometer: XevoG2-XS Q-Tof, Waters; ultra-high performance liquid chromatography: UPLC (Acquity UPLC I-Class), Waters).

[0157] As shown in Figure 10A, the rhGH-Lys-azido-140 sample contained a component 26 Da smaller than the theoretical molecular weight (22,265 Da). This component is presumably the product of reduction of the terminal azide moiety (-N3) of Lys-azido to (-NH2). This clearly indicates that the azide group of the commonly used unnatural amino acid Lys-azido is unstable when inserted into proteins, and is prone to reduction to form reduction products, resulting in a loss of coupling activity. As shown in Figure 10B, the rhGH-NBOK-140 sample did not contain a significant amount of impurities, demonstrating the greater stability of the recombinant protein produced from NBOK compared to Lys-azido.

[0158] (2) Comparison of coupling efficiency [ka] As shown in the reaction scheme above, 30kD BCN-PEG (prepared in-house based on Chinese Patent CN112279906A) was site-specifically coupled to rhGH-Lys-azido-140 via a click reaction (where R1 to R2 corresponds to the N- to C-terminus of the amino acid sequence). The target protein obtained as described above was adjusted to 0.5mg / ml with PBS buffer at pH 7.0. The BCN-PEG solid was added to the rhGH-Lys-azido solution at molar ratios of 1:15 and 1:25 (recombinant protein to 30kD BCN-PEG), respectively, and dissolved by thorough shaking to obtain a clear, transparent solution. The reaction mixture was then sealed and allowed to react while shaking on a thermostatic shaker (25°C, 70 rpm). Samples were taken at predetermined intervals, and the reaction results were analyzed using SDS-PAGE. The reaction was terminated after 72 hours, with a conversion rate of approximately 50%-70%. The reaction results are shown in FIG. 11A.

[0159] Referring to Example 8, 30 kD aminooxy PEG was site-specifically coupled to rhGH-NBOK-140 via oximation. Sampling was performed at predetermined intervals, and the reaction results were analyzed using SDS-PAGE. The reaction was terminated after 48 hours, with a conversion rate of approximately 90%. The reaction results are shown in Figure 11B.

[0160] Combining the molecular weight measurement results from high-resolution mass spectrometry, it can be concluded that the reduction of Lys-azido prevented the product from coupling with BCN-PEG, resulting in a decreased coupling rate. On the other hand, the recombinant protein containing unnatural amino acids of the present invention showed significantly higher conversion rates when coupled with PEG than the recombinant protein containing Lys-azido, and the reaction time was significantly shorter, thereby significantly improving the reaction efficiency.

[0161] Unless otherwise specified, all terms used herein have the meanings that are commonly understood by those skilled in the art.

[0162] The embodiments described in the present invention are merely illustrative and do not limit the scope of protection of the present invention. Those skilled in the art can make various other substitutions, changes and improvements within the scope of the present invention, therefore the present invention is not limited to the above embodiments, but is limited only by the claims.

Claims

1. an unnatural amino acid, The unnatural amino acid is a compound having a structure represented by formula (I-3), formula (I-4), formula (I-5), or formula (I-6), 【Chemical Formula 1】 【Chemistry 2】 【Chemistry 3】 【Chemistry 4】 In the formula, X' represents a C0-C6 linear alkylene group, or X' represents a C0-C4 linear alkylene group, one of which is -CH ２ - can be optionally replaced by -O- or -NH-; The R １ , R ２ Each independently represents hydrogen, Unnatural amino acids.

2. The unnatural amino acid is a compound having any one of the following structures: 【Chemistry 5】 The unnatural amino acid of claim 1 .

3. 3. Use of the unnatural amino acid of claim 1 or 2, The use is in the production of a recombinant protein, a recombinant protein conjugate, a recombinant human growth hormone, or a conjugate of recombinant human growth hormone and polyethylene glycol.

4. A recombinant protein, characterized in that the unnatural amino acid of claim 1 or 2 is arranged at at least one site in the amino acid sequence.

5. The carbonyl terminal group of the unnatural amino acid in the recombinant protein of claim 4 and "NH ２ A recombinant protein conjugate, characterized in that the "-O-" terminal group-containing coupling moiety is formed by forming an oxime bond.

6. The recombinant protein is recombinant human growth hormone, and the "NH ２ The "-O-" end group-containing coupling moiety is "NH ２ -O-" end group containing polyethylene glycol, The recombinant protein conjugate of claim 5.

7. Said “NH ２ The —O—” end group-containing coupling moiety is polyethylene glycol having a molecular weight of 10 to 100 KD; The recombinant protein conjugate of claim 6.

8. The amino acid sequence of the recombinant human growth hormone is as shown in SEQ ID NO:

1. The recombinant protein conjugate of claim 6.

9. an unnatural amino acid is arranged at position 107 of the amino acid sequence SEQ ID NO: 1; The recombinant protein conjugate of claim 8.

10. Use of the recombinant protein conjugate described in any one of claims 5 to 9 in the manufacture of a drug for treating growth and development disorders caused by insufficient secretion of endogenous growth hormone, a drug for treating growth and development disorders caused by Turner syndrome, or a drug for treating adult growth hormone deficiency.

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