JP6099756B2 - Methods for introducing fluorine-containing amino acids into peptides or proteins - Google Patents

Description

The present invention relates to a technique applied to biodistribution measurement of PET diagnostic reagents, antibody-type diagnostic agents, peptide-type diagnostic agents, and protein-type pharmaceutical agents. More specifically, the present invention relates to a method for introducing a fluorine-containing amino acid into a peptide or protein (fluorine introduction method), particularly, a method for introducing an ¹⁸ F-containing amino acid into a peptide or protein ( ¹⁸ F labeling method).

The radioisotope ¹⁸ F (half-life: 110 minutes) of fluorine is known in the clinical field as the most useful nuclide for PET probe applications.

Currently, [ ¹⁸ F] FDG, which is a glucose derivative, is used for cancer diagnosis as an ¹⁸ F-labeled diagnostic agent. This diagnostic agent uses the accumulation of [ ¹⁸ F] FDG in cancer tissue due to high sugar metabolic activity in cancer tissue, and is used for ultra-early diagnosis of cancer and judgment of therapeutic effect. .

  JP-A-2009-106268 (Patent Document 1) discloses FMT (fluoromethyl-L-tyrosine) and FET (fluoroethyl-L-tyrosine), which are fluorine-containing amino acids, in one-pot using an enzymatic method. A method of introducing into the N-terminus of a peptide or protein (Nexta method) is described.

JP 2009-106268 A

Although ¹⁸ F-labeled diagnostic agents that utilize sugar metabolic activity are used clinically, some may not be applied depending on the cancer diagnosis. On the other hand, the use of PET in diagnosis of diseases other than cancer is also highly expected. For this reason, many probes for radioactive diagnostic agents other than [ ¹⁸ F] FDG have been proposed for use in ultra-early diagnosis of cancer, cranial nerve disease and cardiovascular disease diagnosis. However, in order to synthesize these various kinds of new probes, a conventional automatic synthesizer must be designed and manufactured for each probe, which is extremely inefficient. This inefficiency has hindered the development of new probes.

Conventionally, [ ¹⁸ F] SFB (N-succinimidyl-4- [ ¹⁸ F] fluorobenzoate) has been widely used as a reagent for labeling peptides, proteins and other macromolecules with ¹⁸ F. However, when batch synthesis is performed using [ ¹⁸ F] SFB, the synthesis time requires as long as 1 to 1.5 hours. Also, the operation is complicated. For this reason, some facilities that can be synthesized using [ ¹⁸ F] SFB have been limited. Furthermore, since ¹⁸ F labeling sites cannot be selectively designated and the activity of peptides and proteins may be impaired, peptides and proteins capable of ¹⁸ F labeling have been limited.

On the other hand, the method of introducing an ¹⁸ F-labeled amino acid into a peptide or protein is preferable because it does not impair the function of the peptide or protein to be labeled. Examples of the technique for introducing ¹⁸ F-labeled amino acid include peptide synthesis, genetic engineering, and enzymatic chemistry. However, the peptide synthesis method has a problem that it takes one day or more to introduce one amino acid molecule, the genetic engineering method is troublesome, and the enzyme chemical method has a problem that the introduction efficiency is not so good.

  For example, according to the Nexta method (enzymatic method) disclosed in Japanese Patent Application Laid-Open No. 2009-106268, 1) a target peptide or protein, 2) a fluorine-containing amino acid to be introduced, 3) tRNA, 4) an aminoacyl tRNA synthetase And 5) The fluorine-containing amino acid is introduced into the target peptide or protein using a mixed solution containing an aminoacyl-tRNA protein transferase (claim 1). Aminoacylation of tRNA, transfer reaction of amino acid to the target peptide or protein occurs subsequently, and aminoacyl tRNA is produced spontaneously in the system, and tRNA acts as a catalyst and is reused ([0010]). This method discloses that the reaction is completed in 20 minutes ([0031]).

However, when the fluorine-containing amino acid to be introduced is an [ ¹⁸ F] -containing amino acid, there is a problem in using the conventional Nexta method. That is, first, it takes about 20 to 30 minutes to prepare [ ¹⁸ F] -containing amino acid (synthesis, purification, radiochemical purity test, etc.). Subsequently, when the prepared [ ¹⁸ F] -containing amino acid is subjected to the conventional Nexta method, the Next method itself requires 20 minutes. Therefore, in order to introduce an ¹⁸ F-labeled amino acid into a peptide or protein, a total time of 40 to It takes about 50 minutes. In this case, it is necessary to reduce the time of the Nexta method, judging from the half-life of the radioisotope ¹⁸ F being 110 minutes.

Accordingly, an object of the present invention is to provide a method capable of introducing a fluorine-containing amino acid into a peptide or protein in a shorter time than a conventional method using an enzymatic chemical method. In particular, an object of the present invention is to provide a method capable of introducing an [ ¹⁸ F] -containing amino acid into a peptide or protein in a shorter time than a conventional method using an enzymatic chemical method.

  As a result of intensive studies, the present inventors have previously formed a complex of a peptide or protein into which a fluorine-containing amino acid is to be introduced and an aminoacyl-tRNA protein transferase, and then performing a Nexta-like reaction, It has been found that the time for introducing fluorine-containing amino acids can be greatly reduced, and the present invention has been completed.

  The present invention includes the following inventions.

(1) mixing a peptide or protein into which a fluorine-containing amino acid is to be introduced and an aminoacyl tRNA protein transferase to form a complex of the peptide or protein and the aminoacyl tRNA protein transferase;
Mixing the complex with a reagent solution containing a fluorine-containing amino acid to be introduced, tRNA, and aminoacyl-tRNA synthetase, and introducing the fluorine-containing amino acid into the peptide or protein. A method for introducing a fluorine-containing amino acid.

(2) The method according to (1) above, wherein in the complex formation step, an excess molar amount of the peptide or protein is used with respect to the aminoacyl-tRNA protein transferase.

(3) In the step of forming the complex, the peptide or protein is used in an amount of 2/1 to 10,000 / 1, expressed in molar ratio with respect to the aminoacyl-tRNA protein transferase. ) Or the method according to (2).

(4) The method according to any one of (1) to (3) above, wherein the aminoacyl tRNA protein transferase is leucyl / phenylalanyl tRNA protein transferase (L / F-T).

(5) the fluorine-containing amino acids include ^[18 F], The method according to any one of the above (1) to (4).

(6) The fluorine-containing amino acids are 2-amino-3- (4- [ ¹⁸ F] fluoromethoxyphenyl) propionic acid ([ ¹⁸ F] FMT) and 2-amino-3- [4- (2- [ ¹⁸ The method according to any one of (1) to (5) above, which is selected from the group consisting of [F] fluoroethoxy) phenyl] propionic acid ([ ¹⁸ F] FET).

(7) The N-terminal residue of the peptide or protein to which the fluorine-containing amino acid is to be introduced is a basic amino acid residue, and the fluorine-containing amino acid is introduced to the N-terminus of the peptide or protein. The method according to any one of (6).

  In the present invention, “peptide” refers to a peptide having an amino acid sequence containing two or more amino acids. In the present invention, “protein” refers to a peptide having a molecular weight of 5,000 or more. Furthermore, the protein in the present invention is a peptide that can exhibit some function / activity in vivo, for example, when it is produced in vivo.

  The “amino acid” in the present invention includes α-, β-, and γ-amino acids.

  In the present invention, “introducing fluorine” is performed by introducing a fluorine-containing amino acid. Therefore, in this specification, “fluorine introduction” is used in the same meaning as introduction of a fluorine-containing amino acid.

Furthermore, in the present invention, in order to more efficiently introduce a fluorine-containing amino acid into a peptide or protein using an enzymatic method, the following (2-1) to (2-10) may be used. That is, in the above method, the efficiency of introducing the ¹⁸ F-labeled amino acid is improved by adding a specific sequence to the peptide or protein to which the ¹⁸ F-labeled amino acid is to be introduced in advance to obtain an activated peptide or protein. Introduction of the activation sequence is an optional step.

(2-1)
A basic amino acid residue as the N-terminal amino acid residue, an amino acid residue adjacent to the N-terminal amino acid residue, an amino acid residue having a hydroxyl group in the side chain, a hydrophilic amino acid residue having a positive charge in the side chain, Alternatively, an activation sequence having at least an amino acid residue whose side chain is —H, —CH ₃ or —C (CH ₃ ) C ₂ H ₅ is added to a peptide or protein to which fluorine is to be introduced, and the activation peptide Or obtaining a protein;
Mixing the obtained activated peptide or protein and aminoacyl-tRNA protein transferase to form a complex of the peptide or protein and the aminoacyl-tRNA protein transferase;
Mixing the complex with a reagent solution containing a fluorine-containing amino acid to be introduced, tRNA, and aminoacyl-tRNA synthetase, and introducing the fluorine-containing amino acid into the peptide or protein. A method for introducing a fluorine-containing amino acid.

(2-2)
The method according to (2-1) above, wherein the N-terminal residue of the peptide or protein into which fluorine is to be introduced is not a basic amino acid residue.

(2-3)
The second amino acid residue from the N-terminus of the peptide or protein into which fluorine is to be introduced is an amino acid residue having a hydroxyl group in the side chain, a hydrophilic amino acid residue having a positive charge in the side chain, and a side chain of -H , —CH ₃ , —C (CH ₃ ) C ₂ H ₅ and —CH ₂ CONH _2, which is not any of the amino acid residues, the method of (2-2) above.

(2-4)
The method according to any one of (2-1) to (2-3) above, wherein the amino acid having a hydroxyl group in the activation sequence is serine or threonine.

(2-5)
The method according to any one of (2-1) to (2-4) above, wherein the hydrophilic amino acid residue having a positive charge in the activation sequence is lysine or arginine.

(2-6)
The method according to any one of (2-1) to (2-3) above, wherein the second amino acid residue in the activation sequence is glycine, alanine, isoleucine, tyrosine or asparagine.

(2-7)
The method according to any one of (2-1) to (2-6) above, wherein the activation sequence contains a spacer group.

(2-8)
The method according to (2-7) above, wherein the spacer group is a peptide.

(2-9)
The method according to (2-7) above, wherein the spacer group is an alkylene oxide-containing group having 2 to 6 carbon atoms.

(2-10)
The method according to any one of (2-1) to (2-9) above, wherein the basic amino acid residue is a lysine residue or an arginine residue.

In the present invention, a complex of a peptide or protein to which a fluorine-containing amino acid is to be introduced and an aminoacyl-tRNA protein transferase is formed in advance, and then a reaction similar to the Nexta method is performed, whereby the reaction of introducing the fluorine-containing amino acid is Time can be greatly shortened (for example, time of 1 minute or less). Thus, the method of the present invention is very useful for introducing [ ¹⁸ F] -containing amino acids into peptides or proteins, judging from the half-life of radioisotope ¹⁸ F being 110 minutes.

In Example 1, the measurement result of fluorescence detection HPLC in the initial 0 minute, incubation 1 minute, and incubation 10 minutes is shown.

[1. Protein or peptide to which fluorine is to be introduced]
The peptide or protein into which fluorine is to be introduced does not matter whether it has a natural amino acid sequence or a non-natural amino acid sequence. A peptide or protein having a natural amino acid sequence can be obtained, for example, by isolation from various living organisms. Alternatively, it may be artificially produced using a known technique such as a genetic engineering technique or organic synthesis. A peptide or protein having an unnatural amino acid sequence can also be artificially produced using a known technique such as a genetic engineering technique or organic synthesis.

  The sequence of the peptide or protein to which fluorine is to be introduced is not particularly limited, but the N-terminal residue is a basic amino acid residue (for example, lysine residue or arginine residue) for the progress of the Nexta method.

  However, [2. As described in [Activation Sequence], prior to complex formation between the peptide or protein and aminoacyl-tRNA protein transferase, an activation sequence is added to the peptide or protein into which fluorine is to be introduced in advance to activate. It may be a peptide or a protein.

[2. Activation sequence]
The peptide or protein into which fluorine is to be introduced is optionally converted into an activated peptide or protein by adding an activation sequence prior to complex formation (before fluorine introduction).

  The activation sequence includes at least a peptide sequence consisting of two specific amino acid residues. Each of the specific 2 amino acid residues may be a residue derived from a natural amino acid or a residue derived from an unnatural amino acid.

  The N-terminal amino acid residue of the activation sequence is a basic amino acid. Specific examples include lysine residues and arginine residues.

Examples of the amino acid residue adjacent to the N-terminal amino acid residue of the activation sequence (that is, the second amino acid residue from the N-terminal) include the following.
-Amino acid residue having a hydroxyl group in the side chain (hydroxyl group includes normal hydroxyl group and phenolic hydroxyl group)
-A hydrophilic amino acid residue having a positive charge in the side chain-Amino acid residue in which the side chain is -H, -CH ₃ , -C (CH ₃ ) C ₂ H ₅ or -CH ₂ CONH ₂

  Among these amino acid residues, in view of the high activation efficiency of the fluorine introduction reaction, amino acid residues having a normal hydroxyl group (not a phenolic hydroxyl group) in the side chain and hydrophilicity having a positive charge in the side chain Amino acid residues are more preferred.

Examples of amino acids having a hydroxyl group in the side chain include serine, threonine, and tyrosine. Examples of hydrophilic amino acids having a positive charge in the side chain include lysine and arginine. An example of an amino acid whose side chain is —H is glycine, an example of an amino acid whose side chain is —CH ₃ is alanine, and an example of an amino acid whose side chain is —C (CH ₃ ) C ₂ H ₅ is isoleucine. An example of an amino acid whose side chain is —CH ₂ CONH ₂ is asparagine.

  When the second amino residue is a natural α-amino acid, the activation efficiency should be approximately in the order of serine, arginine, threonine, lysine, glycine, alanine, isoleucine, tyrosine and asparagine. Has been confirmed by the present inventors.

  The activation sequence can include a spacer group attached to the second amino acid residue. The spacer group can give the activation sequence a structure that preferably matches the substrate binding pocket of the enzyme used in the fluorine-containing amino acid introduction step described below.

  The spacer group may be a peptide chain or a structure other than that. Examples of structures other than peptides include divalent organic groups. Examples of the divalent organic group include an alkylene oxide-containing group. The alkylene oxide in the alkylene oxide-containing group may be an alkylene oxide having 2 to 6 carbon atoms, preferably ethylene oxide or propylene oxide. The divalent organic group may be a polyalkylene oxide-containing group having these as a repeating unit. The spacer group may be appropriately labeled with an isotope or a fluorescent label by those skilled in the art according to various measurement methods and detection methods combined with the present invention.

[3. Addition of activation sequence]
The activation sequence is optionally added to the peptide or protein into which fluorine is to be introduced. Preferably, the activation sequence is added to the N-terminus of the peptide or protein into which fluorine is to be introduced. Thereby, an activated peptide or protein is obtained.

  When the activation sequence consists only of the N-terminal amino acid residue and the amino acid residue adjacent thereto (the second amino acid residue from the N-terminal), the second amino acid residue is a peptide or protein into which fluorine is to be introduced. To join. If the activation sequence has a spacer group, the spacer group binds to the peptide or protein into which fluorine is to be introduced.

  The method for adding the activation sequence can be appropriately selected by those skilled in the art depending on the binding mode between the activation sequence and the peptide or protein into which fluorine is to be introduced.

  For example, when the binding mode is a peptide bond, a normal peptide synthesis method can be used. More specifically, there is a peptide solid phase synthesis method. A polystyrene polymer gel bead with a diameter of about 0.1 mm whose surface is modified with an amino group is used as the solid phase, and amino acid chains are extended one by one through a dehydration reaction. When the sequence of the target peptide is completed, it is cut out from the solid surface to obtain the target substance. In addition, in order to change the N terminus of a protein to an arbitrary sequence, it can be rearranged into an arbitrary sequence using an engineered gene at the stage of protein synthesis using E. coli.

  If the binding mode is not a peptide bond (eg PEG chain), use an active ester group (succinimide) or the like at the end of the target linker and react with the N-terminal amino acid of the peptide / protein. .

[4. Formation of complex]
A peptide or protein into which a fluorine-containing amino acid is to be introduced and an aminoacyl-tRNA protein transferase are mixed to form a complex of the peptide or protein and the aminoacyl-tRNA protein transferase. When the activation sequence is arbitrarily introduced, the obtained activation peptide or protein and aminoacyl tRNA protein transferase are mixed to form a complex of the peptide or protein and the aminoacyl tRNA protein transferase. .

  An aminoacyl-tRNA protein synthase is an enzyme that transfers an amino acid added to the 3 'end of tRNA to the amino terminus (N-terminus) of the protein and catalyzes a peptide bond formation reaction without passing through the genetic code. The aminoacyl tRNA protein transferase may be a wild type or a mutant type having the same function as the wild type. The aminoacyl-tRNA protein transferase can be prepared by appropriately synthesizing by a person skilled in the art according to a known method (for example, a genetic engineering technique) or by purchasing a commercially available product.

  As a specific example of the aminoacyl tRNA protein transferase, leucyl / phenylalanyl tRNA protein transferase (L / F transferase) can be used. L / F transferase is derived from E. coli and is an enzyme that catalyzes the reaction of transferring hydrophobic amino acids such as phenylalanine, leucine, and methionine bound to tRNA to peptides or proteins having lysine or arginine at the N-terminus. is there.

In the complex formation step, an excess molar amount of the peptide or protein is used with respect to the aminoacyl-tRNA protein transferase. For example, the peptide or protein (P) is expressed in molar ratio (P / T) to the aminoacyl tRNA protein transferase (T), for example, in an amount of 2/1 to 10,000 / 1. The amount is preferably 10/1 to 2,000 / 1, more preferably 50/1 to 500/1. By using an excess molar amount of the peptide or protein relative to the aminoacyl tRNA protein transferase, almost all the aminoacyl tRNA protein transferase is converted into a complex with the peptide or protein. By forming a complex in advance and then performing a Nexta-like reaction, the reaction cycle is efficiently performed, and the time for introducing the fluorine-containing amino acid into the complex is greatly reduced (for example, 1 minute) The following time) There is a great advantage when the fluorine-containing amino acid is an [ ¹⁸ F] -containing amino acid.

[5. Amino acid having fluorine]
An amino acid having fluorine is a molecule in which fluorine is introduced into the basic skeleton of amino acid (a structure having an amino group and a carboxyl group in the same molecule). For example, a molecule in which fluorine is introduced into a natural or unnatural amino acid as a basic skeleton by fluorine substitution or by labeling with a fluorine-containing group can be mentioned. The fluorine nuclide is not particularly limited, but the radioisotope ¹⁸ F is particularly preferable because it is useful in applications to PET probes.

  The fluorine-containing amino acid is allowed to have a label or modification other than fluorine as appropriate. Examples of labels include stable isotope labels and fluorescent labels. In this case, measurement and detection can be performed based on a mass value based on a stable isotope and a signal based on a fluorescent label. Examples of modification include modification with an enzyme substrate and an antigenic substance. In this case, detection and purification can be performed based on an enzyme chemical technique or an enzyme immunoscience technique.

Specific examples of amino acids having fluorine include the following.
2-amino-3- (2,3,4,5,6-pentafluorophenyl) propionic acid (ie pentafluorophenylalanine)
2-amino-3- (4-fluoromethoxyphenyl) propionic acid (ie fluoromethyltyrosine)
2-amino-3- [4- (2-fluoroethoxy) phenyl] propionic acid (ie fluoroethyltyrosine)

Among these, in the present invention, 2-amino-3- (4-fluoromethoxyphenyl) propionic acid and 2-amino-3- [4- (2-fluoroethoxy) phenyl] propionic acid are preferable, and their radioactivity is preferred. 2-amino-3- (4- [ ¹⁸ F] fluoromethoxyphenyl) propionic acid and 2-amino-3- [4- (2- [ ¹⁸ F] fluoroethoxy) phenyl] propionic acid, which are isotope labels, More preferred. The amino acid having fluorine is appropriately selected according to aminoacyl tRNA synthetase and aminoacyl tRNA protein transferase described later.

[6. Introduction of amino acids having fluorine]
A fluorine-containing amino acid is introduced into the complex of the peptide or protein (or optionally activated peptide or protein) and the aminoacyl-tRNA protein transferase. At this time, a reagent solution containing tRNA and aminoacyl tRNA synthetase (aaRS) is used.

tRNA can be converted to aminoacyl-tRNA by aminoacylation by aminoacyl-tRNA synthetase, and the aminoacyl group of aminoacyl-tRNA can be converted into a peptide or a peptide by aminoacyl-tRNA protein transferase (in the present invention, the complex) Any material can be used as long as it can be transferred to a protein, whether natural or non-natural. Therefore, it is not limited whether the tRNA structure has a cloverleaf secondary structure, and the size thereof is not limited. The tRNA can be prepared by appropriate synthesis by a person skilled in the art according to a known method, or by purchasing a commercially available product. Specific examples of tRNA include tRNA ^Phe and tRNA ^Leu .

  An aminoacyl-tRNA synthetase (aaRS) is an enzyme responsible for synthesizing an aminoacyl-tRNA that is generally a substrate in translation of the genetic code in a ribosome. The aaRS may be a wild type or a mutant type. The aaRS can be prepared by appropriately synthesizing by a person skilled in the art according to a known method (for example, genetic engineering technique), or by purchasing a commercially available product.

  There are 20 types of wild-type aaRS having substrate specificity for each of the 20 amino acids used to synthesize proteins. For example, aaRS corresponding to lysine is called lysyl tRNA synthetase. In the present invention, aaRS corresponding to the amino acid basic skeleton in the fluorine-containing amino acid to be introduced (that is, having substrate specificity for the amino acid basic skeleton) can be used.

  As the mutant aaRS, an aaRS mutant in which a mutation occurs such that the substrate specificity for a fluorine-containing amino acid is higher than the substrate specificity for a fluorine-free amino acid is used. For example, E. coli-derived phenylalanyl tRNA synthetase mutant (Ala294 → Gly) (Chembiochem, 2002, 02-03, which has a higher substrate specificity for fluoromethyltyrosine and fluoroethyltyrosine than that for phenylalanine) 235-237) can be used.

In the reagent solution, a buffer solution suitable for the introduction reaction of the fluorine-containing amino acid, salts, reducing agent, polyamine, energy source and the like can be appropriately contained. Examples of the buffer include Hepes buffer and Tris-acetic acid. Examples of the salts include magnesium salts and potassium salts. Examples of the reducing agent include DTT (dithiothreitol). Examples of the polyamine include spermidine. Examples of the energy source include adenosine triphosphate. To give a more specific example, the composition at the final concentration is 10 mM MgCl ₂ , 1 mM Spermidine, 50 mM Hepes buffer (pH 7.6), and the composition at the final concentration is 2.5 mM ATP, 20 mM. KCl, 2 mM DTT B solution can be mixed and used.

The concentration of each component in the mixed solution of the peptide or protein (or optionally activated peptide or protein) and the aminoacyl-tRNA protein transferase complex and the reagent solution can be arbitrarily set. it can.
For example, the molar ratio of the peptide or protein complex and tRNA in the mixed solution is 30: 1 to 1: 1, more preferably 10: 1 to 4: 1. In the present invention, since tRNA is reused in the system, the amount used may be small. The molar ratio of the peptide or protein complex and aminoacyl-tRNA synthetase in the mixed solution is 100: 1 to 2: 1, more preferably 50: 1 to 20: 1. The molar ratio of the peptide or protein complex and the fluorine-containing amino acid in the mixed solution is 100: 1 to 1: 1000, more preferably 25: 1 to 1: 400, still more preferably 1: 1 to 1: 120. It is. While the fluorine-containing amino acid can be introduced even in a small amount compared to the peptide or protein complex, it can inhibit the reaction system even in an excessive amount compared to the peptide or protein complex. It is not considered. Therefore, when using a valuable fluorine-containing amino acid such as a radioisotope fluorine-containing amino acid, the amount of fluorine-containing amino acid used can be kept small compared to the peptide or protein complex. When the peptide or protein complex is more valuable, the amount of the activated peptide or protein complex used can be reduced to a small amount with respect to the fluorine-containing amino acid. Since a complex of the peptide or protein (optionally activated peptide or protein) and the aminoacyl-tRNA protein transferase is formed in advance, the reaction of introducing a fluorine-containing amino acid into the complex is very fast.

Any reaction conditions such as temperature and pH can be selected according to the enzyme used. The reaction temperature is preferably 0 ° C to 50 ° C, more preferably 4 ° C to 37 ° C. The reaction pH is preferably 6-9, more preferably 7-8. A reaction time of about 10 minutes is sufficient, and the reaction is completed in about 1 minute. There is a great advantage when the fluorine-containing amino acid is an [ ¹⁸ F] -containing amino acid.

  In introducing an amino acid having fluorine, a complex of the peptide or protein (or optionally activated peptide or protein) and the aminoacyl tRNA protein transferase, the fluorine-containing amino acid to be introduced, tRNA, and aminoacyl A reaction solution in which a reagent solution containing tRNA synthetase is mixed is prepared. Each component may be mixed at once or in any order. Moreover, the fluorine-containing amino acid can use 1 type or multiple types.

The reaction solution is subjected to incubation (for example, 37 ° C., 60 minutes), whereby the fluorine-containing amino acid introduction reaction proceeds. When the fluorine-containing amino acid is an [ ¹⁸ F] -containing amino acid, the incubation time should be a short time, for example, about 10 minutes or less, preferably about 5 minutes, more preferably about 1 minute.

  The reaction can be stopped, for example, by mixing a TFA (trifluoroacetic acid) aqueous solution.

  After termination of the reaction, the peptide or protein into which the fluorine-containing amino acid has been introduced by concentration desalting is recovered as the target product. For example, the purified target product can be purified by concentration desalting by ZipTip treatment.

[7. Peptide or protein fluorine introduction kit]
In this invention, the kit which can be used for the method of introduce | transducing fluorine into the above-mentioned peptide or protein is provided. Items include at least tRNA, aminoacyl tRNA synthetase, and aminoacyl tRNA protein transferase. Each item may be provided in a state where it exists in a separate container, or for example, it may be provided in a state where tRNA and aminoacyl tRNA synthetase coexist in one container. The aminoacyl tRNA protein transferase is present in the container by itself. Preferably, one or more fluorine-containing amino acids (for example, phenylalanine and derivatives thereof) are included. Further, the above-described buffer solution, salts, reducing agent, polyamine, energy source, and the like may be included.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to the following examples.

[Example 1]
Lys-Bradykinin was used as a peptide into which a fluorine-containing amino acid was to be introduced.
As the fluorine-containing amino acid, cold 2-amino-3- [4- (2-fluoroethoxy) phenyl] propionic acid (fluoroethyltyrosine: FET) was used.
Leucyl / phenylalanyl tRNA protein transferase (L / F-T) was used as the aminoacyl tRNA protein transferase.

The composition at the final concentration in the amino acid introduction step is
MgCl ₂ 10 mM
Supermidine 1 mM
Hepes buffer (pH 7.6) 50 mM
L / F-T 2 μM ea
Lys-Bradykinin 0.17 mM ea
Then, the mixture was incubated at 37 ° C. for 5 minutes so that all L / FT molecules were converted into L / FT / Lys-Bradykinin complexes (complex solution A).

Separately, the composition at the final concentration in the amino acid introduction step is
MgCl ₂ 10 mM
Supermidine 1 mM
Hepes buffer (pH 7.6) 50 mM
tRNA 6μM
FET 2μM
ARS 2μM
Then, the mixture was incubated at 37 ° C. for 10 minutes to form aminoacyl tRNA (reagent solution B).

  The molar ratio of L / F-T: ARS: FET was 1: 1: 1.

  Reagent solution B was added to complex solution A and incubated at 37 ° C. for 10 minutes to introduce FET into Lys-Bradykinin.

  The reaction efficiency was calculated by fluorescence detection HPLC (manufactured by JASCO Corporation, PU2085) at the initial stage of the reaction (0 minutes, at the time of mixing the complex solution A and the reagent solution B), 1 minute of reaction, and 10 minutes of reaction.

Fluorescence detection conditions: ex. 274 nm em. 300nm
(The reaction efficiency was calculated by detecting the decrease in FET over time.)
-Deployment conditions: 0.1% TFA (0-2 min)
0.1% TFA-AN (0-100%; 2-12 min)

  The measurement results of the fluorescence detection HPLC at each time point of initial 0 minute, incubation 1 minute, and incubation 10 minutes are shown in FIG. Horizontal axis: Retention Time [min], Vertical axis: Intensity.

  From FIG. 1, the peak derived from FET disappeared after 1 minute of incubation, and the peak of FET bound to Lys-Bradykinin was detected. Furthermore, no change was observed in the reaction efficiency even after 10 minutes of incubation. From this, it is estimated that the reaction time for introducing FET into Lys-Bradykinin is 1 minute or less.

Claims (7)

Mixing a peptide or protein into which a fluorine-containing amino acid is to be introduced and an aminoacyl tRNA protein transferase to form a complex of the peptide or protein and the aminoacyl tRNA protein transferase;
Mixing the complex with a reagent solution containing a fluorine-containing amino acid to be introduced, tRNA, and aminoacyl-tRNA synthetase, and introducing the fluorine-containing amino acid into the peptide or protein. A method for introducing a fluorine-containing amino acid.   The method according to claim 1, wherein in the complex formation step, an excess molar amount of the peptide or protein is used with respect to the aminoacyl-tRNA protein transferase.   In the complex formation step, the peptide or protein is used in an amount of 2/1 to 10,000 / 1 expressed in molar ratio with respect to the aminoacyl tRNA protein transferase. The method described.   The method according to any one of claims 1 to 3, wherein the aminoacyl tRNA protein transferase is leucyl / phenylalanyl tRNA protein transferase (L / F-T). The method according to claim 1, wherein the fluorine-containing amino acid comprises [ ¹⁸ F]. Wherein the fluorine-containing amino acid, 2-amino -3- (4- ^[18 F] fluoro-methoxyphenyl) propionic acid ^([18 F] FMT) and 2-amino ^{-3- [4- (2- [18 F} ] fluoro 6. The method according to any one of claims 1 to 5, selected from the group consisting of (ethoxy) phenyl] propionic acid ([ ¹⁸ F] FET).   The N-terminal residue of the peptide or protein to which the fluorine-containing amino acid is to be introduced is a basic amino acid residue, and the fluorine-containing amino acid is introduced to the N-terminus of the peptide or protein. The method described in 1.

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