Abstract:
The present invention provides an  18 F-labeled azide compound usable in the Huisgen reaction which enables  18 F-labeling although only a small quantity of alkyne compound is available as a counterpart substrate, more specifically the  18 F-labeled azide compound enabling the PET to be applied to peptides or oligonucleotides and enabling the  18 F-labeling of any sites of oligonucleotide other than the 5′ end or 3′ end thereof, a reagent for  18 F-labeling, and a method for  18 F-labeling of an alkyne compound using the same.

Description:
FIELD OF THE INVENTION 
     The present invention relates to an  18 F-labeled azide compound, a reagent for  18 F-labeling, and a method for  18 F-labeling of an alkyne compound using the same. The present invention is suitably applicable to production of a radioactive tracer indispensable for positron emission tomography (hereinafter, called “PET”). 
     BACKGROUND OF THE INVENTION 
     PET is a method that includes administering, into a living body, a tracer labeled with a short-term radionuclide which releases positrons such as  18 F or  11 C so that γ rays generated from the tracer is measured by a PET camera (detector comprising a gamma ray scintillator and a photoelectron multiplier tube), and imaging a body distribution of the γ ray by a computer. The PET can non-invasively and quantitatively track down the movement of materials over time in vivo, therefore, is now actively employed as a very useful measurement technique in many different fields such as biology, development of pharmaceutical products, and medical services. 
     Examples of the short-term radionuclide used in the PET are  18 F and  11 C, and compounds labeled with these radionuclides are used as the tracer. There is a very broad application range for the  11 C utilizing carbon atoms present in organic compounds, therefore,  11 C can be considered to be an ideal radionuclide. However, the  11 C has such a short half life as 20 minutes, imposing the restriction that the process from synthesis through PET measurement must be completed within a very short time frame. In contrast, the  18 F having a half life longer than that of the  11 C, 110 minutes, is easy to handle and widely used in, for example,  18 F-labeled glucose. Despite such an advantageous half life, positrons released by the  18 F decrease with time, making the PET measurement similarly difficult. Faced with such a difficulty, a fast and simplified  18 F labeling method is desirably accomplished. 
     To prepare an  18 F-labeled compound, its base material,  18 F, is supplied through nuclide transformation by a cyclotron. More specifically, ions accelerated by the cyclotron are crashed into water including  18 O to invite nuclide transformation from  18 O to  18 F. The  18 F-labeled compound can be theoretically obtained when a very weak water solution including  18 F ions thus obtained and a compound selected as a PET target are reacted with each other. 
     In fact, it is conventionally extremely difficult to directly bond  18 F to the compound for the PET. Therefore, an indirect  18 F labeling method is employed, wherein an intermediate compound labeled with  18 F and having a functional group to be bonded to another compound (intermediate medium thus characterized is hereinafter called “ 18 F prosthetic group”) is prepared in advance and then bonded to the compound for the PET. 
     Examples of properties required for the  18 F prosthetic group are: 1) easily and speedily synthesizable; 2) bondable to a target compound rapidly and efficiently under temperate conditions; and 3) hardly affecting in-vivo kinetics of the target compound. To meet these requirements, an  18 F prosthetic group which leverages the Huisgen reaction is currently attracting attention. 
     Describing the Huisgen reaction, an organic azide compound and alkynes, as a result of a [3+2] cycloaddition reaction generated therebetween, are transformed into a 1,2,3-triasole derivative (see the following chemical formula). 
     
       
                 
         
             
             
         
      
     
     In the case where a terminal alkyne is used as the alkynes, copper (I) ions work as a catalyst, and a 1,4-disubstituted body is selectively obtained. This reaction is not disturbed by any other functional groups, if present, such as alcohol, amine, amide, ester, and halide, therefore, a target material, triaosle, can be obtained at a high yield. The reaction uneventfully advances in any of reaction solvents such as alcohol, conventional organic solvents, and water. The asides and alkynes have a number of advantages; they can be easily introduced in various organic compounds, and they do not produce excessive post-reaction waste matters. Therefore, the asides and alkynes are cited as typical examples which represent the Click Chemistry advocated by K. B. Sharpless et al. who was awarded with Nobel Prize in Chemistry. 
     In the studies conducted in recent years using the Huisgen reaction, azide compounds or alkyne compounds labeled with  18 F are used as the  18 F prosthetic group to introduce the  18 F prosthetic group in any target compounds. Below are given specific examples of such studies. 
     Marik and Sutcliffe, University of California, Davis, fluorinated a tosylate body having an acetylene group on its molecular end in the presence of [ 18 F]KF and kalium scavenger (Kryptofi x222) and purified through distillation so that  18 F-labeled fluoroalkynes are obtained as the  18 F prosthetic group (Non-Patent Document 1). Then, they generated the Huisgen reaction between the  18 F prosthetic group and azidated peptide in the presence of a catalytic system including iodinated copper (I), sodium ascorbate, and di-isopropyl ethylamine as amine base to perform  18 F-labeling of the peptide at room temperature for 10 minutes (see the following reaction formula). 
     
       
                 
         
             
             
         
      
     
     They further performed  18 F-labeling of α v β 6  specific peptide using the  18 F prosthetic group in which n=3 in the above formula, and successfully obtained PET images in mice in vivo (Non-Patent Document 2). 
     Another study using the  18 F prosthetic group in which n=1 in the above formula was also reported by a study group of Sungkyunkwan University, Korea (Non-Patent Document 3). 
     A study group representing SIEMENS MEDICAL SOLUTIONS USA, INC. reported the use of a compound comparable to n=0 in the above formula (Patent Document 1), wherein propargyl tosylate is  18 F-transformed to generate an  18 F prosthetic group ([ 18 F]-3-fluoropropyne) in a reaction container and leave the generated  18 F prosthetic group unpurified, and an overly large quantity of azide substrate is added to a reaction solution to generate a coupling reaction using copper (I) acetate. 
     The Non-Patent Document 4 recites an example in which an  18 F-labeled azide compound is used, wherein the Huisgen reaction is generated with oligopeptide having a terminal acetylene group by using [ 18 F] fluoroethylene azide as  18 F so that the oligopeptide is successfully labeled with  18 F (see the following chemical formula). 
     
       
                 
         
             
             
         
      
     
     A study group of Inha University, Korea reported the use of a plurality of  18 F-labeled alkyne compounds and  18 F-labeled azide compounds (see the following chemical formula) as the  18 F prosthetic group, wherein they performed  18 F-labeling of a variety of substrates (see the Non-Patent Document 5). However, their method directly performing the coupling reaction without purifying the  18 F prosthetic group similarly uses an overly large quantity of compounds to be  18 F-labeled as the  18 F prosthetic group. 
     
       
                 
         
             
             
         
      
     
     A study group of Stanford University purified the  18 F prosthetic group by employing preparative HPLC more easily automatable than distillation to thereby obtain an  18 F-labeled alkyne compound having a high purity (Non-Patent Document 6). They generated the Huisgen reaction using a catalytic system including copper sulfate—sodium ascorbate but no amine base to label ligands of integrin α v β 3  with  18 F and captured PET images in mice. 
     A study group of Dresden-Rossendor Research Center, Germany purified 4-[ 18 F]Fluoro-N-(prop-2-ynyl)benzamide synthesized by the following method by employing SPE (solid phase extraction) and used the purified material as the  18 F prosthetic group, and then performed the  18 F-labeling of azidated Neurotensin (8-13) peptide through the Huisgen reaction (Non-Patent Document 7). 
     
       
                 
         
             
             
         
      
     
     They discussed the conditions of the Huisgen reaction to reduce the quantity of peptide used in each  18 F-labeling test, and found out that an optimal condition is to use the catalytic system including copper sulfate and sodium ascorbate with a borate buffer, and finally succeeded in reducing the quantity of azidated Neurotensin (8-13) peptide to 1 mg (approximately 1 μmol). However, it is necessary to further reduce the quantity for practical use. 
     A study group of TRIUMF, Canada, based on their hypothesis that aryl fluorine is probably more metabolically stable than aliphatic fluorine, developed an aryl fluorine  18 F prosthetic group synthesizable in one stage, which was reported in 2008 (Non-Patent Document 8). 
     More specifically describing their study, a position 2 nitro group or trimethyl ammonio group of the pyridine derivative expressed by the following chemical formula is fluorinated and purified by the HPLC so that an  18 F prosthetic group having a high purity is obtained, and the  18 F prosthetic group was subjected to the Huisgen reaction with a compound to be  18 F-labeled (azidated peptide precursor) in the presence of a catalytic system including TBTA, Cu (CH 3 CN) 4 PF 6  and di-isopropyl ethylamine. The quantity of the compound to be  18 F-labeled used in their study was, however, 1,400 nmol. This is still a large quantity which needs to be reduced. 
     
       
                 
         
             
             
         
      
     
     In recent years, the gene therapy based on such phenomena as antisense, antigene, decoy, and RNA interference is increasingly progressing. There are ongoing approaches for the gene therapy using natural nucleic acids (DNA, RNA) or artificial nucleic acids having better pharmacological kinetics and physiological activities than natural nucleic acids (DNA, RNA) (for example, 2′-0-MeRNA, phosphorothioate oligo, BNAs, LNA). When it succeeds to label the oligonucleotide of any natural or artificial nucleic acid with  18 F and administer the  18 F-labeled oligonucleotide to a human or an experimental animal as a PET probe to measure intra-body kinetics using a PET camera, studies and researches of oligonucleotides are expected to further advance, accelerating the development of pharmaceutical products in which the technique is leveraged. Further, once the “double strand with complementary strand”, which is a characteristic phenomenon of oligonucleotides, can be observed in vivo by using the  18 F-labeled oligonucleotide (in vivo hybridization), it is facilitated to measure an in-vivo mRNA expression level. This technique is broadly applicable to diagnostics of diseases, and studies and researches of medicine and pharmacy. To meet the needs described so far, the oligonucleotide  18 F-labeling method was so far often studied and reported (for example, Non-Patent Documents 9 to 14). 
     PRIOR ART DOCUMENTS 
     Patent Document 
     
         
         Patent Document 1: WO2006/116629 
       
    
     Non-Patent Documents 
     
         
         Non-Patent Document 1: Marik, J.; Sutcliffe, J. L. Tetrahedron Lett. 2006, 47, 6681-6684. 
         Non-Patent Document 2: Hausner, S. H.; Marik, J.; Gagnon, M. K. J.; Sutcliffe, J. L. J. Med. Chem. 2008, 51, 5901-5904. 
         Non-Patent Document 3: Kim, D. H.; Choe, Y. S.; Jung, K. -H.; Lee, K. -H.; Choi, J. Y.; Choi, Y.; Kim, B. -T. Arch. Pharm. Res. 2008, 31, 587-593. 
         Non-Patent Document 4: Glaser, M.; Arstad, E. Bioconjugate Chem. 2007, 18, 989-993. 
         Non-Patent Document 5: Sirion, U.; Kim, H. J.; Lee, J. H.; Seo, J. W.; Lee, B. S.; Lee, S. J.; Oh, S. J.; Chi, D. Y. Tetrahedron Lett. 2007, 48, 3953-3957. 
         Non-Patent Document 6: Li, Z. B.; Wu, Z.; Chen, K.; Chin, F. T.; Chen, X. Bioconjugate Chem. 2007, 18, 1987-1994. 
         Non-Patent Document 7 Ramenda, T.; Bergmann, R.; Wuest, F. Lett. Drug Des. Discovery 2007, 4, 279-285. 
         Non-Patent Document 8 Inkster, J. A. H.; Guerin, B.; Ruth. T. J.; Adam, M. J. J. Labelled Compd. Radiopharm. 2008, 51, 444-452. 
         Non-Patent Document 9: Dolle, F.; Hinnen, F.; Vaufrey, F.; Tavitian, B.; Crouzel, C. J. Labelled Compd. Radiopharm. 1997, 39, 319-330. 
         Non-Patent Document 10: Kuhnast, B.; Dolle, F.; Terrazzino, T.; Rousseau, B.; Loc&#39;h, C.; Vaufrey, F.; Hinnen, F.; Doignon, I.; Pillon, F.; David, C.; Crouzel, C.; Tavitian, B. Bioconjugate Chem. 2000, 11, 627-636. 
         Non-Patent Document 11: Kuhnast, B.; de Bruin, B.; Hinnen, F.; Tavitian, B.; Dolle, F. Bioconjugate Chem. 2004, 15, 617-627. 
         Non-Patent Document 12: Hedberg, E.; Langstrom, B. Acta Chem. Scand. 1997, 51, 1236-1240. 
         Non-Patent Document 13: Hedberg, E.; Langstrom, B. Acta Chem. Scand. 1998, 52, 1034-1039. 
         Non-Patent Document 14: Christopher, W. L.; VanBrocklin, H. F.; Taylor, S. E. J. Label. Compd. Radiopharm. 2002, 45, 257-268. 
       
    
     SUMMARY OF THE INVENTION 
     Problem to be Solved by the Invention 
     As described so far, variously different  18 F-labeled azide compounds and  18 F-labeled alkyne compounds were synthesized to be used as the  18 F prosthetic group to perform the  18 F-labeling through the Huisgen reaction. Further, kinetics of the  18 F-labeled compounds were studied by the PET. However, it was always necessary to use a large quantity of acetylene group-modified substrates as a counterpart of the reaction in the case of the  18 F-labeled azide compounds, and it was always necessary to use a large quantity of azide group-modified substrates as a counterpart of the reaction in the case of the  18 F-labeled alkyne compounds probably because of a reason described below. 
     The Huisgen reaction per se is received in the field of organic chemistry as an easy-to-handle reaction that can rapidly and easily advance when an azide compound and alkyne are simply mixed with each other in the presence of a copper (I) catalyst. However, the Huisgen reaction used in combination with the  18 F-labeling, which is totally different to the Huisgen reaction alone, is very difficult to perform because a cyclotron can only produce a very small quantity of  18 F, leaving no choice but to use a very low-concentrated  18 F prosthetic group. According to the reaction kinetics, a reaction rate is calculated from the function of a reaction rate constant and a reaction substrate. When the low-concentrated  18 F prosthetic group is used for the Huisgen reaction, therefore, it is necessary to choose any reaction system having a large reaction rate constant or increase the concentration of any compound as a labeling target. However, none of the conventional reaction systems was designed to have a substantially large reaction rate constant. Under the circumstances, there was no option but to increase the concentration of any compound to be labeled. Therefore, the quantity of any compound to be labeled was inevitably increased. In the case where the compound to be labeled is such a material that requires considerable time and cost when isolated from natural products or variously synthesized in a large quantity such as oligopeptides or oligonucleotides, only a limited number of PET images may be captured, however, any full-scale PET studies using a number of sequences of oligopeptides or oligonucleotides are still difficult. So far, there is no known example in which oligonucleotides were  18 F-labeled through the Huisgen reaction. 
     The Non-Patent Documents 9 to 14 report the oligonucleotide  18 F-labeling studies without using the Huisgen reaction. Employing any of the methods reported in these documents for performing the PET, it is still necessary to prepare a large quantity of oligonucleotide precursors that requires considerable time and cost for obtaining a large quantity and a wide variety. Therefore, the reported methods were not applicable to practical use. Further, these methods can only perform the  18 F-labeling of the 5′ end or 3′ end of oligonucleotide, while failing to perform the  18 F-labeling of any other sites thereof. These facts resulted in a small quantity of oligonucleotide precursors that can be used, leading to a strong demand for an  18 F-labeling method that allows a large number of options for parts to be labeled. 
     The present invention was accomplished to solve the conventional problems. The present invention provides an  18 F-labeled azide compound usable in the Huisgen reaction which enables  18 F-labeling although only a small quantity of alkyne compound is available as a counterpart substrate, more specifically the  18 F-labeled azide compound enabling the PET to be applied to peptides or oligonucleotides and enabling the  18 F-labeling of any sites of oligonucleotide other than the 5′ end or 3′ end thereof, a reagent for  18 F-labeling, and a method for  18 F-labeling of an alkyne compound using the same. 
     Means for Solving the Problem 
     To solve the conventional problems, the inventors of the present invention discussed preferable designs of an  18 F-labeled azide compound when applied to the PET. Prior to the  18 F-labeling, a compound having a small molecular weight is preferably selected to minimize any impact on in vivo kinetics of a compound subjected to the PET. An ideal example of the compound in this regard could be  18 FCH 2 N 3 . However, this material having a low boiling point is easily gasified, therefore, practically unusable to avoid the risk of radiation exposure. Therefore, the compound to be selected preferably has a relatively high boiling point. The compound to be selected is desirably capable of absorbing strong UV that can be detected by a UV detector of a high-performance liquid chromatography (hereinafter, called “HPLC”) apparatus to facilitate post-synthesis purification and concentration, and designed to have such a boiling point that melted fractions will not be volatilized when concentrated. Further, the compound is desirably synthesizable in one stage from a precursor of the  18 F-labeled azide compound and chemically stable. 
     A candidate of the compound meeting the required properties adduced by the inventors of the present invention is a phenyl azide compound labeled with  18 F. Because of a difficulty in converting hydrogen of a phenyl group into fluorine, the inventors discussed introduction of a fluoroalkyl group in the phenyl group and tried to figure out a design that allows a smallest fluoromethyl group to be introduced. The inventors synthesized a compound which conforms to the design, and performed the Huisgen reaction between the compound and acetylene group-modified oligonucleotide. Then, they found out that their approach solves the conventional problems, and finally completed the present invention. 
     An  18 F-labeled azide compound according to the present invention is expressed by the following structural formula 1). 
     
       
                 
         
             
             
         
      
     
     The  18 F-labeled azide compound has an azide group and thereby generates the Huisgen reaction with a compound having a carbon-carbon triple bond. Because of the structural characteristic, the  18 F-labeled azide compound can be used as a reagent for  18 F-labeling. 
     According to the finding by the inventors of the present invention, the  18 F-labeled azide compound can be transformed speedily and easily into an  18 F-labeled triasole derivative by the Huisgen reaction with an alkyne compound in the presence of a copper compound catalyst. As compared to the Huisgen reaction using the conventional  18 F-labeled azide compound, this reaction advances although the alkyne compound, which is a counterpart substrate, is low-concentrated. Therefore, this reaction is applicable to the  18 F-labeling of peptides and oligonucleotides characterized in difficulty of preparing a large quantity of substrates. 
     The reaction is preferably performed in the presence of ascorbate and an amine-based base by using a mixed solvent including water and a water-soluble organic solvent as s reaction solvent. Examples of the water-soluble organic solvent are; diphenyl sulfone, dimethyl sulfoxide, benzophenone, tetrahydrothiophene-1,1-dioxide, N,N-dimethylacetamide, N-methyl-2-pyrrolidinone, N-methyl-ε-caprolactam, 1,3-dimethyl-2-imidazolidinone, methanol, ethanol, n-propanol, i-propanol, n-butanol, i-butanol and t-butanol. Of these examples, dimethylsulfoxide (hereinafter, called DMSO) is particularly a suitable example because of its remarkable resolving power, good safety, less corrosiveness, good handleability because of its moderate melting point and boiling point, and good yield. Examples of the amine-based base are; diisopropyl ethylamine, triethylamine, pyridine, 2,6-lutidine, and (tris((1-benzyl-1H-1,2,3-triasole-4-yl)methyl)amine. Of these examples, (tris((1-benzyl-1H-1,2,3-triasole-4-yl)methyl)amine (hereinafter, called “TBTA”) is a particularly preferable example. 
     The alkyne compound used in an alkyne compound  18 F-labeling method according to the present invention can be a nucleotide derivative. An oligonucleotide is a biological molecule applicable to pharmaceutical products because of its physiological activity based on such phenomena as antisense, antigene, decoy, and RNA interference. Further, there are ongoing studies of artificial nucleic acids having better pharmacological kinetics and physiological activities than natural nucleic acids (DNA, RNA) (for example, 2′-0-MeRNA, phosphorothioate oligo, BNAs, LNA). When it succeeds to label the oligonucleotide of any natural or artificial nucleic acid with  18 F and administer the  18 F-labeled oligonucleotide to a human or an experimental animal as a PET probe to measure intra-body kinetics using a PET camera, studies and researches of oligonucleotides are expected to further advance, accelerating the development of pharmaceutical products in which the technique is leveraged. 
     The alkynyl group-modified oligonucleotide enables the  18 F-labeling of not only the 5′ end or 3′ end of oligonucleotide but also any other sites thereof. Thus, any sites of oligonucleotide other than the 5′ end or 3′ end thereof can be successfully  18 F-labeled. 
    
    
     DESCRIPTION OF EMBODIMENTS 
     Hereinafter, examples of the present invention are described in detail. 
     Synthesis of 4-methyl benzenesulfonic acid 4-azide benzyl (3a) 
     First, 4-methyl benzenesulfonic acid 4-azide benzyl (3a), which is used as a precursor of the  18 F-labeled azide compound according to the present invention, was synthesized by the method expressed by the following reaction formula. 
     
       
                 
         
             
             
         
      
     
     4-azidebenzyl alcohol (2a) (prepared by the method recited in (Andersena, J. et al.; Synlett 2005, 14, 2209-2213) (149 mg, 1.00 mmol) was dissolved in methylene chloride (5.0 mL), and pyridine (0.162 mL, 2.00 mmol) and p-toluenesulfonic acid (343 mg, 1.05 mmol) were added thereto at 0° C. After the mixed matter was agitated for 30 minutes, water is added thereto, and the resulting solution was quenched. An aqueous layer of the solution was removed therefrom, and an organic layer thereof was washed sequentially with 1 mol/L hydrochloric acid, saturated sodium acid carbonate water solution, and saturated salt solution and then dried with anhydrous sodium sulfate. Then, the solvent was distilled away under a reduced pressure. A residue thereby obtained was mixed with diethyl ether (3 mL) and then filtered through a plug of cotton to remove any insoluble matters therefrom. The filtered solution was mixed with hexane (5 mL) and agitated, and then left at rest for 30 minutes so that crystals were deposited. A supernatant liquid thereby obtained was removed from the solution by means of a pipette. The crystals were washed with hexane and then dried under a reduced pressure so that 4-methyl benzenesulfonic acid 4-azide benzyl (3a) (218 mg, 0.719 mmol, 71.9%) was obtained in the form of colorless crystals. Below are recited  1 H-NMR spectrum,  13 C-NMR spectrum, and mass spectrum (EI) of the obtained material. 
       1 H-NMR (400 MHz, CDCl 3 ) 
     δ: 2.45 (3H, s), 5.02 (2H, s), 6.97 (2H, d, J=8.3 Hz), 7.24 (3H, d, J=8.3 Hz), 7.34 (2H, d, J=8.3 Hz), 7.79 (2H, d, J=8.3 Hz) 
       13 C-NMR (100 MHz, CDCl 3 ) 
     δ: 21.7, 71.3, 119.2, 127.9, 129.9, 129.9, 130.3, 133.3, 141.0, 144.9 
     HRMS (EI) 
     calc 303.0677 obs 303.0650 
     Synthesis of 4-methyl benzenesulfonic acid 3-azide benzyl (3b) 
     In a manner similar to the description given earlier, 4-methyl benzenesulfonic acid 3-azide benzyl (3b), which is an isomer of the compound (3a), was obtained by the yield of 51.8%. 
     
       
                 
         
             
             
         
      
     
     Below are recited IR spectrum,  1 H-NMR spectrum,  13 C-NMR spectrum, mass spectrum (EI), and element analysis of the obtained material. 
     IR (film, KBr) 
     2114, 1593, 1489, 1452, 1360, 1292, 1177, 945, 835, 814, 781, 665 cm −1    
       1 H-NMR (400 MHz, CDCl 3 ) 
     δ: 2.45 (3H, s), 5.03 (2H, s), 6.85 (1H, s), 6.97 (1H, d, J=7.8 Hz), 7.03 (1H, d, J=7.8 Hz), 7.30 (1H, t, J=7.8 Hz), 7.33 (2H, d, J=8.5 Hz), 7.79 (2H, d, J=8.5 Hz) 
       13 C-NMR (100 MHz, CDCl 3 ) 
     δ: 21.6, 71.0, 118.8, 119.5, 124.7, 128.0, 129.9, 130.1, 133.1, 135.3, 140.5, 145.0 
     LRMS (EI) 
     calc 303 obs 303 
     Element Analysis 
     calc. H, 4.32%; C, 55.43%; N, 13.85%. 
     obs. H, 4.32%; C, 55.59%; N, 13.89%. 
     Synthesis of Acetylene Group-Modified Oligonucleotide 
     Synthesis of Acetylene Group-Modified Oligonucleotide (4a) 
     Next, an acetylene group-modified oligonucleotide (4a) (sequence in the formula presents natural DNA) was synthesized by the method recited in the following literature (S. Obika, et al. Bioorganic and Medicinal Chemistry Letters, 2009, 19, 3316-3319) as an alkyne compound used as the substrate of the Huisgen reaction. 
     
       
                 
         
             
             
         
      
     
     Synthesis of Acetylene Group-Modified Oligonucleotide (4b) 
     Then, an acetylene group-modified oligonucleotide precursor (4b) (underscored part in the following chemical formula represents 2′,4′-BNA, and other parts represent natural DNA, “s” in the formula represent a phosphorothioate bond) having 2′,4′-BNA (also called LNA, see the following chemical formula) and a phosphorothioate bond was synthesized as an artificial nucleic acid by the method recited in the following literature (S. Obika, et al. Bioorganic and Medicinal Chemistry Letters, 2009, 19, 3316-3319). 
     
       
                 
         
             
             
         
      
     
     Synthesis of Acetylene Group-Modified Oligonucleotide (4b) 
     Further, an acetylene group-modified oligonucleotide precursor (4c) (underscored part in the following chemical formula represents 2′,4′-BNA, and other parts represent natural DNA) in which 2′ and 4′ positions of deoxyribose are cross-linked was synthesized by the method recited in the following literature (S. Obika, et al. Bioorganic and Medicinal Chemistry Letters, in press, oi:10.1016/j.bmcl.2009.04.063). 
     
       
                 
         
             
             
         
      
     
     Synthesis of  18 F-Labeled Azide Compound and  18 F-Labeling of Acetylene Group-Modified Oligonucleotide 
     Then, the  18 F-labeled azide compound was synthesized by means of the 4-methyl benzenesulfonic acid-4-azide benzyl (3a) and 4-methyl benzenesulfonic acid-3-azide benzyl (3b) synthesized earlier. Further, a coupling reaction was performed between the  18 F-labeled azide compound and the acetylene group-modified oligonucleotide through the Huisgen reaction. 
     Example 1 
     In an example 1, a tosylate group of the 4-methyl benzenesulfonic acid 4-azide benzyl (3a) was substituted with  18 F to synthesize an  18 F-labeled azide compound (1a). Then, the coupling reaction was performed between the compound and the acetylene group-modified oligonucleotide (4a) through the Huisgen reaction. Hereinafter, the example 1 is described in detail. 
     
       
                 
         
             
             
         
      
     
     Synthesis of  18 F-Labeled Azide Compound (1a) 
     The [ 18 O] water (approximately 2 mL, supplied by TAIYO NIPPON SANSO CORPORATION) was irradiated by a 12-meV electronic beam (HM-12S supplied by Sumitomo Heavy Industries, Ltd., current value: 50 μA, for 30 minutes) so that [ 18 F] fluorine ions were generated. An approximately 50-GBq fluoroion [ 18 O] water solution thus obtained was guided in a labeling synthesis apparatus (GNMS-α, supplied by GNMS-ALPHA DAINIPPON SEIKI CO., LTD.) installed in a hot cell, and let through an anion-exchange resin cartridge (SAIKA-SPE SAX-30, supplied by AiSTI SCIENCE). The [ 18 F] fluorine ions adsorbed thereto were removed by carbonic acid hydrogenion tetra N-butyl ammonium (0.025 mol/L, 80% acetonitrile/water solution, 0.6 mL), and washed with 0.6 mL of acetonitrile. Then, the [ 18 F] fluoroion solution was guided in a first reaction container and heated to 110° C. to be dried and solidified under a reduced pressure in helium flow, and further azeotropically dried with acetonitrile (1 mL). A residual thereby obtained is mixed with an acetonitrile (1 mL) solution of 4-methyl benzenesulfonic acid 4-azide benzyl (1a) (6.0 mg) and subjected to a reaction at 85° C. for five minutes. A radiochemical yield obtained by analysis at the time was 99%. The reaction mixture was isolated and purified by semi-preparative HPLC (conditions: COSMOSIL MS-11, 10×250 mm column supplied by NAKALAI TESQUE INC., dipped in 40% acetonitrile/water for six minutes and then dipped in 60% acetonitrile/water for 14 minutes, flow rate: 1 mL/min., target material was eluted after retained for 15 to 16 minutes). 
       18 F-Labeling of Acetylene Group-Modified Oligonucleotide (4a) Using  18 F-Labeled Azide Compound (1a) 
     All the fractions including the  18 F-labeled azide compound (1a) thus isolated and purified were gathered and transferred to a second reaction container already containing therein 0.18 mL of DMSO. Under a reduced pressure in helium flow while being heated to 40° C., acetonitrile was carefully volatilized. To the DMSO water-mixed solution (about 0.5 mL) of the  18 F-labeled azide compound (1a) thus obtained was added a buffer (100 mmol/L sodium biphosphate buffer, pH: 7.0, 60 μL), acetylene group-modified oligonucleotide (4a) (0.50 mmol/L water solution, 40 μL), copper sulfate (50 mmol/L water solution, 12 μL), TBTA (tris(1-benzyl-1H-1,2,3-triasole-4-yl)methyl)amine, 50 mmol/L DMSO solution (6 μL), and sodium ascorbate (50 mmol/L water solution, 12 μL) to generate a reaction at 40° C. for 15 minutes. A radiochemical yield obtained by analysis at the time was 92%. The reaction mixture was diluted with water (0.3 mL), and then isolated and purified by semi-preparative HPLC (conditions: COSMOSIL AR-11, 10×250 mm column supplied by NAKALAI TESQUE INC., column temperature: 50° C., linearly gradient in a 10-20% CH3CN/0.1 mol/L TEAA buffer for 20 minutes, flow rate: 4 mL/min., target material was eluted after retained for 14 to 15 minutes). All the fractions including targeted [ 18 F] (5a) were gathered, and acetonitrile was evaporated under a reduced pressure, so that a TEAA buffer solution (5 mL) of  18 F-labeled oligonucleotide (5a) was obtained. 
     Time for and Yield of Synthesis of  18 F-Labeled Oligonucleotide (5a) 
     A length of time required for synthesizing the  18 F-labeled oligonucleotide (5a) and a yield thereof was; synthesis time: 84 minutes, radiation of isolated (5a): 2.53 GBq, specific radioactivity: 2366 GBq/μmol, chemical purity (UV 260 nm): 95%, radiation chemical purity: 87%, radiochemical yield based on [ 18 F] fluorine ions: 5.2% (not decay-compensation), and 8.6% (decay-compensated). 
     The synthesizing method can desalinate or concentrate TEAA if necessary. Below are described in detail desalination steps. 
     The TEAA buffer solution of [ 18 F] (5a) was let through Sep-Pak Plus C18 (supplied by Nihon Waters K.K., preconditioned with 40 mL of EtOH and 40 mL of water) and washed with water (5 mL) twice, and then dried for one minute in nitrogen gas flow so that [ 18 F] (5a) was eluted with ethanol (1 mL). Then, the ethanol was volatilized in nitrogen gas flow to obtain a concentrated [ 18 F] (5a) water solution. This water solution was diluted with a suitable volume of physiological salt solution so that a solution to be administered to animals was prepared. Although the process time further increased by 30 minutes, 90% was recovered (decay-compensated). 
     Example 2 
     Similarly to the synthesis in the example 1, a tosylate group of the 4-methyl benzenesulfonic acid-3-azide benzyl (3b) was substituted with  18 F to synthesize an  18 F-labeled azide compound (1b). Then, the coupling reaction was performed between the compound and the acetylene group-modified oligonucleotide (4a) through the Huisgen reaction. 
     Although not described in the examples because it is explicitly known from the technical common knowledge, an  18 F-labeled ortho-azide compound can be obtained through an operation performed similarly to the example 1 by using 2-azidebenzyl alcohol in place of 4-azidebenzyl alcohol (2a) used as a parent material in the example 1. 
     
       
                 
         
             
             
         
      
     
     Synthesis of  18 F-Labeled Azide Compound (1b) 
     Similarly to the synthesis of the  18 F-labeled azide compound (1a) in the example 1, the  18 F-labeled azide compound (1b) was synthesized. 
     Labeling of Acetylene Group-Modified Oligonucleotide (4a) Using  18 F-Labeled Azide Compound (1b) 
     Similarly to the method described in the example 1, the acetylene group-modified oligonucleotide (4a) was labeled by using the  18 F-labeled azide compound (1b). The coupling reaction was performed between the  18 F-labeled azide compound (1b) and the acetylene group-modified oligonucleotide (4a) through the Huisgen reaction, so that a TEAA buffer solution (5 mL) of  18 F-labeled oligonucleotide (5b) was obtained. 
     Time for and Yield of Synthesis of  18 F-Labeled Oligonucleotide (5b) 
     A length of time required for synthesizing the  18 F-labeled oligonucleotide (5b) and a yield thereof were; synthesis time: 83 minutes, radiation of isolated (5b): 2.12 GBq, specific radioactivity: 1809 GBq/μmol, chemical purity (UV 260 nm): 99%, radiation chemical purity: 93%, radiochemical yield based on [ 18 F] fluorine ions: 4.2% (not decay-compensation), and 7.2% (decay-compensated). 
     Example 3 
     In an example 3, similarly to the synthesis in the example 2, a tosylate group of the 4-methyl benzenesulfonic acid-3-azide benzyl (3b) was substituted with  18 F to synthesize the  18 F-labeled azide compound (1b). Then, the coupling reaction was performed between the compound and the acetylene group-modified oligonucleotide (4b) through the Huisgen reaction. 
     
       
                 
         
             
             
         
      
     
       18 F-Labeling of Acetylene Group-Modified Oligonucleotide (4b) 
     The coupling reaction was performed between the  18 F-labeled azide compound (1b) thus synthesized and the acetylene group-modified oligonucleotide (4b) through the Huisgen reaction similarly to the method in the example 2, so that an  18 F-labeled oligonucleotide (5c) was obtained. 
     Time for and Yield of Synthesis of  18 F-Labeled Oligonucleotide (5c) 
     A length of time required for synthesizing the  18 F-labeled oligonucleotide (5c) and a yield thereof were; synthesis time: 95 minutes, radiation of isolated (5c): 0.862 GBq, specific radioactivity: 762 GBq/μmol, chemical purity (UV 260 nm): 96%, radiation chemical purity: &gt;99%, radiochemical yield based on [ 18 F] fluorine ions: 1.7% (not decay-compensation), and 3.1% (decay-compensated). 
     Example 4 
     In an example 4, similarly to the synthesis in the example 2, a tosylate group of the 4-methyl benzenesulfonic acid-3-azide benzyl (3b) was substituted with  18 F to synthesize the  18 F-labeled azide compound (1b). Then, the coupling reaction was performed between the compound and the acetylene group-modified oligonucleotide (4c) through the Huisgen reaction. 
     
       
                 
         
             
             
         
      
     
       18 F-Labeling of Acetylene Group-Modified Oligonucleotide (4c) 
     The coupling reaction was performed between the  18 F-labeled azide compound (1b) thus synthesized and the acetylene group-modified oligonucleotide (4b) through the Huisgen reaction, so that an  18 F-labeled oligonucleotide (5d) was obtained. 
     Time for and Yield of Synthesis of  18 F-Labeled Oligonucleotide (5d) 
     A length of time required for synthesizing the  18 F-labeled oligonucleotide (5d) and a yield thereof were; synthesis time: 83 minutes, radiation of isolated (5c): 1.66 GBq, specific radioactivity: 3205 GBq/μmol, chemical purity (UV 260 nm): 98%, radiation chemical purity: 96%, radiochemical yield based on [ 18 F] fluorine ions: 3.3% (not decay-compensation), and 5.6% (decay-compensated). 
     Comparison to the Prior Art 
     It was confirmed form the results of the examples 1 to 3 that the alkyne compound can be  18 F-labeled at a high yield when the Huisgen reaction is performed between the  18 F-labeled azide compounds (1a) and (1b) which are very dilute and the acetylene group-modified oligonucleotides which are very dilute in the presence of the copper compound catalyst. Table 1 shows required quantities and concentrations of compounds to be  18 F-labeled in the conventional  18 F-labeling method and the  18 F-labeling method according to the present invention. As compared to the conventional  18 F-labeling method, the  18 F-labeling using the  18 F-labeled azide compounds (1a) and (1b) according to the examples 1 to 3 can be accomplished although the required quantities and concentrations of the compounds to be  18 F-labeled are significantly small as is clearly known from the table. 
                                                   TABLE 1                       quantity of   concentration           compound to be   of compound to             18 F-labeled   be  18 F-labeled           (nmol)   (μmol/L)                                    oligonucleotide   method recited in the Non-patent Document 9   100-200               method recited in the Non-patent Document 10   200-400   200-400           method recited in the Non-patent Document 11   141-275   353-688           method recited in the Non-patent Document 12   70   778           method recited in the Non-patent Document 13    52-140    520-1040           method recited in the Non-patent Document 14    76-600    688-5503       Huisgen reaction   method recited in the Non-patent Document 1   300   136           method recited in the Non-patent Document 2   400   533           method recited in the Non-patent Document 3   48700   37462           method recited in the Non-patent Document 4   3400   17000           method recited in the Non-patent Document 5   15000   30000           method recited in the Non-patent Document 6   678   565           method recited in the Non-patent Document 7   1000   12500           method recited in the Non-patent Document 8   1400   9333             18 F-labeled azide compounds (1a) and (1b)   10-20   10-40               The Non-Patent Documents 1 to 8 recite the  18 F-labeling methods using the Huisgen reaction, whereas the Non-Patent Documents 9 to 14 recite the conventional methods wherein oligonucleotide is  18 F-labeled.            
Reaction Conditions
 
     To perform the  18 F-labeling through the Huisgen reaction using the  18 F-labeled azide compound according to the present invention, the synthesis is preferably performed remotely by using a mechanical device to avoid exposure to radiation. To serve the purpose, it is desirable to use a labeling synthesis apparatus in a shielded draft. As a result, it becomes necessary to meet within a limited amount of time such a need to obtain a trace level of 18F-labeled azide compound having a very high purity from large quantities of precursors, resolved matters, and different reagents. Moreover, the Huisgen reaction per se should speed up and achieve a high yield. To meet all of these needs, optimal reaction conditions were thoroughly discussed. 
     Optimal Conditions for Huisgen Reaction 
     To efficiently perfrom the Huisgen reaction, the inventors focused on a reaction rate and discussed optimal conditions that allow the reaction speed to be as high as possible because the present invention synthesizes only a small quantity of  18 F-labeled azide compound, making it indispensable to find such conditions that two very dilute substrates can be reacted under moderate conditions. 
     The inventors discussed the reaction conditions by using a non-labeled  19 F azide compound as the substrate of the alkyne compound, a model of which is N-(prop-2-ynyl)benzamide. First, the high-concentrated substrates were subjected to the Huisgen reaction for screening of organic solvents most suitable for the reaction. 1 mM/L copper sulfate was used as a catalyst, 2 mM/L sodium ascorbate was used as a reducer, 1 mM/L TBTA was used in the Huisgen reaction. Further, the organic solvents were variously changed at the capacity ratio of water to organic solvent=3 to 7. 
     
       
                 
         
             
             
         
      
     
     As a result, it was learnt that the addition of the non-protic solvent improved the yield, and the DMSO particularly improved the yield as illustrated in Table 2. Further, it was clearly known from the comparison of the entries 5 and 6 that the addition of TBTA dramatically improved the yield. 
     
       
         
               
               
               
             
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 entry 
                 solvent a)   
                 yield (%) b)   
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 dioxane 
                 94 
               
               
                 2 
                 DMF 
                 82 
               
               
                 3 
                 CH3CN 
                 1 
               
               
                 4 
                 t-BuOH 
                 95 
               
               
                 5 
                 DMSO 
                 97 
               
               
                 6 
                 DMSO c)   
                 8 
               
               
                   
               
               
                   a) organic solvent:H 2 O = (7:3) 
               
               
                   b) yield obtained from HPLC 
               
               
                   c) TBTA not added 
               
             
          
         
       
     
     The inventors further looked into the reaction conditions in the foregoing reaction when a dilute solution is used. Similarly to concentrations conventionally used in radiation labeling tests, they changed the reaction conditions using a more dilute substrate (100 μmol/L N-(prop-2-ynyl)benzamide and 50 μmol/L non-labeled  19 F azide compound (1b)). Then, it was learnt that the yield improved when the TBTA concentration was equal to or higher than 100 μM/L, and the yield was better when (TBTA concentration)/(Cu concentration) was ½ than 1. Thus, it was confirmed that the ratio less than 1 could favorably improve the yield. Further, the yield is better when the reaction temperature is set to 40° C. than room temperature. 
     
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                 entry 
                 Na Asc 
                 TBTA 
                 Cu 
                 temperature 
                 yield (%) a)   
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 1000 
                 1000 
                 1000 
                 room temperature 
                 71 
               
               
                 2 
                 1000 
                 500 
                 1000 
                 room temperature 
                 79 
               
               
                 3 
                 500 
                 250 
                 500 
                 room temperature 
                 81 
               
               
                 4 
                 200 
                 100 
                 200 
                 room temperature 
                 67 
               
               
                 5 
                 100 
                 50 
                 100 
                 room temperature 
                 41 
               
               
                 6 
                 1000 
                 500 
                 1000 
                 40° C. 
                 94 
               
               
                 7 
                 500 
                 250 
                 500 
                 40° C. 
                 87 
               
               
                   
               
               
                   a) yield obtained from HPLC 
               
             
          
         
       
         
         reaction time: 15 minutes 
         reaction solvent: DMSO/H 2 O=20/80, 10 mmol/L, sodium phosphate buffer pH7 
       
    
     Under the conditions of the entry 2 in Table 3, the yield when pH of the sodium phosphate buffer is variously changed was checked. As illustrated in  FIG. 4 , the yield showed remarkable improvements when pH was 6.5 to 7.5, and marked the highest result when pH was around 7. 
     
       
         
               
               
               
             
               
               
               
             
           
               
                   
                 TABLE 4 
               
               
                   
                   
               
               
                   
                 pH 
                 yield (%) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 6.5 
                 67 
               
               
                   
                 7 
                 79 
               
               
                   
                 7.5 
                 68 
               
               
                   
                   
               
             
          
         
       
     
     Under the conditions of the entry 2 in Table 3, the yield when the DMSO concentration is variously changed was checked. As illustrated in  FIG. 5 , the yield showed remarkable improvements when the DMSO volume percent is at least 30%. 
     
       
         
               
               
               
             
           
               
                   
                 TABLE 5 
               
               
                   
                   
               
               
                   
                 DMSO (volume %) 
                 yield (%) 
               
               
                   
                   
               
             
             
               
                   
                 10 
                 82 
               
               
                   
                 20 
                 79 
               
               
                   
                 30 
                 92 
               
               
                   
                 40 
                 90 
               
               
                   
                 50 
                 92 
               
               
                   
                   
               
             
          
         
       
     
     PET Images 
     The  18 F-labeled oligonucleotide (5a) obtained in the example 1 was administered to a rat, and a PET image was obtained. More specifically, a physiological salt solution containing 45 MBq of the  18 F-labeled oligonucleotide (5a) is administered to an SD rat (8 weeks old, weight: 252 g) under anesthesia though a caudal vein, and an image of the rat was obtained by MicroPET Focus-220, PET apparatus for animals supplied by SIEMENS.  FIG. 1  shows a whole-body image of the rat showing 30-minute accumulation after 120 minutes passed since the probe administration (Maximum Intensity Projection image). The image teaches that the administered  18 F-labeled oligonucleotide (5a) was promptly metabolized in blood and excreted in urine. It is read from the image the deposition of intensive radiation in the bone texture of the whole body, which is, however, considered to result from F-ions generated as the  18 F-labeled oligonucleotide (5a) is metabolized. 
     The  18 F-labeled oligonucleotides (5b), (5c), and (5d) were similarly administered to rats, and PET images of the rats were obtained.  FIGS. 2 to 4  show whole-body images of the rats showing 30-minute accumulation after 120 minutes passed since the administration (Maximum Intensity Projection image). These drawings teach that the 2′,4′-BNA-transformed (5d), because its metabolic rate in blood slows down, is deposited in their kidneys and urinary bladders, and the radioactivity in their bone textures is deteriorated, and further teaches that the 2′,4′-BNA-transformed (5c) having its skeleton bonded to phosphorothioate is metabolized at even a lower metabolic rate and deposited in the kidneys and urinary bladders, and the radioactivity in the bone textures is further deteriorated. 
     As described so far, when the  18 F-labeled oligonucleotide according to the present invention is used, the effects of artificial nucleic acids, such as 2′,4′-BNA-transformed oligonucleotide and phosphorothioate oligonucleotide, can be directly observed. Thus, the present invention is advantageous in that, for example, nuclease tolerance and distribution to tissues can be directly observed in vivo in the studies of the gene therapy using RNA interference. Therefore, the present invention can be leveraged as a technically advantageous tool in the research and development of oligonucleotide pharmaceutical products. 
     The present invention is not necessarily limited to the exemplary embodiment and examples described so far. The present invention includes various modified embodiments as far as they stay within the Scope of Claims and can be easily assumed by the ordinarily skilled in the art. 
     BRIEF DESCRIPTION OF THE DRAWINGS 
       FIG. 1  is a whole-body image (MIP image) of an SD rat showing 30-minute accumulation after 120 minutes passed since an  18 F-labeled oligonucleotide (5a) is administered to the SD rat though a caudal vein. 
       FIG. 2  is a whole-body image (MIP image) of a SD rat showing 30-minute accumulation after 120 minutes passed since an  18 F-labeled oligonucleotide (5b) is administered to the SD rat though a caudal vein. 
       FIG. 3  is a whole-body image (MIP image) of a SD rat showing 30-minute accumulation after 120 minutes passed since an  18 F-labeled oligonucleotide (5c) is administered to the SD rat though a caudal vein. 
       FIG. 4  is a whole-body image (MIP image) of a SD rat showing 30-minute accumulation after 120 minutes passed since an  18 F-labeled oligonucleotide (5d) is administered to the SD rat though a caudal vein. 
     INDUSTRIAL APPLICABILITY 
     The present invention can provide an advantageous means in the field of medicine where nucleic acid oligomer is used, for example, RNA drug development expected to be launched in individualized medicine.