JP2013198426A - Rna detective reagent, examination method, and reagent for synthesizing the rna detective reagent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new RNA detective reagent capable of selectively detecting an RNA strand as a target, and to provide a reagent for synthesizing such an RNA detective reagent.SOLUTION: The new RNA detective reagent to be used comprises: an oligonucleotide having a sequence complementary to RNA as a target; a divalent linker unit with its one end condensed with the 5'-terminated hydroxy group of the oligonucleotide; a fluorescent coloring unit bound to the other end of the divalent linker unit; and a boronic acid unit bound to the fluorescent coloring unit and represented by general formula (1).

Description

  The present invention relates to a reagent for RNA detection, a test method, and a reagent for synthesizing a reagent for RNA detection.

  Genome analysis begins with determining the base sequence of the DNA (deoxyribonucleic acid) molecules that make up the genome. However, it is not possible to obtain information such as where and what gene is present, and where in the cell a specific gene is expressed, only from the data of the base sequence. Therefore, analysis of gene products such as messenger RNA (mRNA; RNA is an abbreviation of ribonucleic acid) transcribed from DNA and proteins produced by translating this, and the nucleotide sequence between species Analysis of the genome proceeds based on comparisons of how similar they are, and further on data on individual genes analyzed by various approaches using experimental organisms such as E. coli and budding yeast.

  Genetic information of DNA in the cell nucleus is transcribed into RNA. Usually, the transcribed RNA moves into the cytoplasm and is translated into protein. Although it is already known that various biopolymers are involved in the regulation of such transcription and translation, recently, RNA that is not translated into protein (also referred to as non-coding RNA; ncRNA) includes these regulation. It has been found that it plays a major role in gene regulation. ncRNA is a general term for RNA that is not translated into protein, and examples include transfer RNA (tRNA), ribosomal RNA (rRNA), micro RNA (miRNA), and small interfering RNA (siRNA).

  In recent years, evidence has been obtained that complex regulation of transcription and translation is carried out in cells of higher organisms such as humans. These also point out the possibility that RNA is used in a wider area than previously thought in biology, especially in gene regulation. For example, the RNAi phenomenon found by Fire and Mellow et al. And caused by a type of ncRNA is found in many organisms from nematodes to humans, and plays an important role in controlling other genes. . At present, the gene knockout method using this RNAi phenomenon is widely used in molecular biology research (see Non-Patent Documents 1 and 2).

  Against this background, techniques for detecting RNA have been actively developed for the purpose of exploring the action of various ncRNAs in cells (see, for example, Patent Document 1).

Special table 2007-533324 gazette

Okazaki, Y .; et al. , Nature, 420, 563-573 (2002). Ota, T .; et al. Nat Genet, 36, 40-45 (2004).

  The present invention has been made in view of the above situation, and is for synthesizing a novel RNA detection reagent capable of selectively detecting a target RNA strand and such an RNA detection reagent. It is an object of the present invention to provide a reagent.

  As a result of intensive studies, the present inventors have developed a fluorescent color developing group consisting of a fluorescent coloring group on the 5′-end of an oligonucleotide having a sequence complementary to the target RNA via a linker unit as a binding group. When a compound in which a boronic acid unit having a unit and a tertiary amino group and a phenylboronic acid group is bound is used as a reagent for RNA detection, when the target RNA is present in the solution, It was found that the fluorescence from the reagent for RNA detection was enhanced as compared. The present invention has been completed based on the above findings, and provides the following.

(1) The present invention provides an oligonucleotide having a sequence complementary to a target RNA, a divalent linker unit in which one end is condensed with a hydroxyl group at the 5′-end of the oligonucleotide, and the divalent linker unit The reagent for RNA detection provided with the fluorescence coloring unit couple | bonded with the other end of this, and the boronic acid unit represented by following General formula (1) couple | bonded with the said fluorescence coloring unit.
(In the general formula (1), R ¹ is a hydrogen atom or an alkyl group, and m and n are each independently an integer of 0 to 4. The boronic acid unit represented by the general formula (1) Is bound to the fluorescent coloring unit by a single bond with a wavy line.)

  (2) The fluorescent coloring unit is preferably a divalent organic group obtained by removing two substituents which may be hydrogen atoms from a condensed polycyclic aromatic compound which may have a hetero atom.

(3) The RNA detection reagent is preferably represented by the following general formula (2).
(In the general formula (2), Nu is a group formed by removing the 5′-terminal hydroxyl group from the oligonucleotide, and X may be a hydrogen atom from a condensed polycyclic aromatic compound which may have a hetero atom. The fluorescent coloring unit that is a divalent organic group obtained by removing two substituents, L is the linker unit that is a divalent organic group, and R ¹ is the same as in the general formula (1). In the above general formula (2), the oxygen atom present between Nu and L means the oxygen atom remaining after removing the hydrogen atom from the 5′-terminal hydroxyl group of the oligonucleotide. )

(4) The RNA detection reagent is preferably represented by the following general formula (3).
(In the above general formula (3), Nu is a group formed by removing the 5′-terminal hydroxyl group from the oligonucleotide, and X may be a hydrogen atom from a condensed polycyclic aromatic compound which may have a hetero atom. In the fluorescent color developing unit, which is a divalent organic group obtained by removing two substituents, R ¹ is the same as in the general formula (1), R ² is a hydrogen atom or an alkyl group, and Z is It is a divalent group or a single bond, and n is an integer of 1 to 18. In the general formula (3), the oxygen atom present between Nu and Z is the 5′- of the oligonucleotide. (This means the oxygen atom remaining after removing the hydrogen atom from the terminal hydroxyl group.)

  (5) Z in the general formula (3) is preferably —P (═O) (— OH) O—.

  (6) The present invention observes whether the fluorescence of the RNA detection reagent is enhanced when the RNA detection reagent is added to the test target and the test target is irradiated with ultraviolet rays or visible light. Thus, it is also an inspection method for determining whether or not the target RNA is included in the inspection object.

(7) This invention is also a reagent for synthesize | combining the reagent for RNA detection represented by following General formula (4).
(In the general formula (4), X is a divalent organic group obtained by removing two substituents which may be hydrogen atoms from a condensed polycyclic aromatic compound which may have a hetero atom, and L is 2 R ¹ is a hydrogen atom or an alkyl group, R ³ and R ⁴ are each independently a boronic acid protecting group, and R ³ and R ⁴ are bonded to each other to form a ring. R ⁵ is a protecting group for the phosphite group, R ⁶ and R ⁷ are each independently an alkyl group, and R ⁶ and R ⁷ may be bonded to each other to form a ring. )

(8) The reagent is preferably represented by the following general formula (5).
(In the general formula (5), X, R ¹ , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ are the same as those in the general formula (4), and R ² is a hydrogen atom or an alkyl group. , N is an integer of 1-18.)

  According to the present invention, a novel RNA detection reagent capable of selectively detecting a target RNA strand and a reagent for synthesizing such an RNA detection reagent are provided.

FIG. 1 is a fluorescence spectrum for (U12) + (B12), (T12) + (B12) and (B12) alone in the example, (a) is a fluorescence spectrum at pH 7.0, (b ) Is a fluorescence spectrum at pH 7.5, and (c) is a fluorescence spectrum at pH 8.0.

  Hereinafter, an embodiment of the reagent for RNA detection of the present invention, a test method using the reagent for RNA detection, and a reagent for synthesizing the reagent for RNA detection will be described. The present invention is not limited to the following embodiments, and can be implemented with appropriate modifications within the scope of the present invention.

  The RNA detection reagent of the present invention comprises an oligonucleotide having a sequence complementary to a target RNA, a divalent linker unit having one end condensed with the 5′-terminal hydroxyl group of the oligonucleotide, and the divalent linker unit. A fluorescent coloring unit bonded to the other end of the linker unit; and a boronic acid unit represented by the following general formula (1) bonded to the fluorescent coloring unit. According to the RNA detection reagent of the present invention, RNA having an arbitrary base sequence can be selectively detected.

(In the general formula (1), R ¹ is a hydrogen atom or an alkyl group, and m and n are each independently an integer of 0 to 4. The boronic acid unit represented by the general formula (1) Is bound to the fluorescent coloring unit by a single bond with a wavy line.)

The reagent for detecting RNA according to the present invention is a fluorescent coloring unit in which a boronic acid unit represented by the above general formula (1) is bound to the 5′-terminal side of an oligonucleotide having a sequence complementary to a target RNA. Is provided. First, in order to explain the action of the reagent for RNA detection of the present invention, a schematic chemical reaction diagram is shown below. In the chemical reaction diagram below, as an example, a case where an anthracene skeleton is used as a fluorescence coloring unit, R ¹ in the general formula (1) is a methyl group, and m and n in the general formula (1) are 1 is shown. Although shown, the present invention is not limited to this.

  The boronic acid unit represented by the general formula (1) includes a phenylboronic acid and a tertiary amino group, and cis-2,3-diol present at the 3′-end of RNA targeted by phenylboronic acid. Form a bond with the structure. However, the bond formation reaction between phenylboronic acid and the 3'-terminal diol of RNA in the solution is an equilibrium reaction largely biased toward the dissociation side, and phenylboronic acid exists alone in the solution containing RNA. Even so, the phenylboronic acid cannot form a bond with RNA. In the RNA detection reagent of the present invention, by arranging the boronic acid unit on the 5′-terminal side of the oligonucleotide having a sequence complementary to the target RNA, the oligonucleotide having the complementary sequence can be obtained. When a double strand is formed with the target RNA and captured, the boronic acid unit is arranged on the 3′-terminal side of the target RNA. This greatly increases the opportunity for the boronic acid unit to form a bond with the 3'-terminal diol of the targeted RNA. That is, the situation is the same as when the target RNA is concentrated around the boronic acid unit, and the equilibrium reaction is greatly inclined toward the bond formation side. On the other hand, even if non-target RNA is present in the solution, this RNA is not captured by the oligonucleotide contained in the RNA detection reagent, so the equilibrium reaction between the boronic acid unit and the RNA is on the dissociation side. It does not bind to phenylboronic acid contained in the boronic acid unit.

  When the boronic acid unit binds to the 3′-terminal side of the target RNA as described above, the RNA detection reagent transmits the fact as fluorescence. Next, this mechanism will be described.

  As described above, the boronic acid unit includes a tertiary amino group, and this tertiary amino group is sandwiched between the phenylboronic acid contained in the boronic acid unit and the fluorescence coloring unit. The electrons of the unshared electron pair contained in the nitrogen atom of the tertiary amino group, when irradiated with excitation light, cause a photochromic electron transfer (PET) phenomenon to occur in the fluorescent coloring unit existing in the vicinity. It moves and the fluorescence from the fluorescence coloring unit disappears. However, as described above, when the phenylboronic acid contained in the boronic acid unit binds to the 3′-end of the target RNA, the PET phenomenon does not occur, and the fluorescence from the fluorescent coloring unit when irradiated with excitation light. Will occur. This will be observed as if the fluorescence was enhanced.

  By such an action, when the target RNA exists, the RNA detection reagent of the present invention can bind to the RNA and transmit the fact as fluorescence. That is, the RNA detection reagent of the present invention enhances the fluorescence when the target RNA is present in the solution when the excitation light is irradiated, and fluoresces when the target RNA is not present in the solution. Do not increase. By the above actions, the RNA detection reagent of the present invention can detect the presence or absence of target RNA.

  An oligonucleotide having a sequence complementary to the target RNA forms a duplex with the target RNA and functions as a recognition site for capturing it. As already mentioned, this recognition site has the function of capturing the target RNA and orienting the cis-2,3-diol structure on the 3′-terminal side of the target RNA toward the boronic acid unit. .

  An oligonucleotide having a sequence complementary to the target RNA is determined based on the base sequence of the target RNA, and an oligonucleotide automatic synthesizer (DNA / RNA) is determined based on the determined base sequence. Synthesized by an automatic synthesizer). Oligonucleotide synthesis by an automated oligonucleotide synthesizer is an established technique, and an oligonucleotide having a sequence complementary to the target RNA in the present invention may be synthesized based on such technique. .

  Needless to say, the complementary oligonucleotides are a combination of the 5′-end side of one oligonucleotide and the 3′-end side of the other oligonucleotide, and the 3′-end of one oligonucleotide. A pair of bases are combined to form a double strand so that one side and the 5′-end side of the other oligonucleotide are combined. Needless to say, the paired bases are bases that are a combination of C (cytosine) and G (guanine), or A (adenine) and U or T (uracil or thymine). If the base sequence of CGAU is present at a certain position of one base sequence, the base sequence GCUA is present at the position corresponding to that of the other base sequence.

  One end of the divalent linker unit is condensed and bound to the hydroxyl group on the 5'-terminal side of the oligonucleotide having a sequence complementary to the target RNA. A fluorescent coloring unit, which will be described later, is bonded to the other end of the linker unit. As a result, the 5'-terminal side of the oligonucleotide having a sequence complementary to the target RNA (that is, the recognition site) and the fluorescence unit are bound to each other via the linker unit.

  The divalent linker unit is not particularly limited. As such a linker unit, an oligonucleotide having a sequence complementary to a target RNA (that is, a recognition site) and a hetero atom having a binding group for binding to a fluorescent coloring unit at both ends may be used. A good carbon chain can be exemplified. In this case, the bonding groups at both ends can be determined independently of each other. Examples of such bonding groups include ether bonds, ester bonds, phosphodiester bonds, amide bonds, carbonate bonds, urethane bonds, alkylene groups, and the like. However, it is not particularly limited.

  A carbon chain that may have a hetero atom is a carbon chain that may have any number of non-carbon atoms in the chain, and the carbon chain includes a single bond, a double bond, and a triple bond. It may be. Moreover, the end of the carbon chain may be a heteroatom. Although it does not specifically limit as number of atoms contained in the carbon chain which may have a hetero atom, About 1-25 pieces can be illustrated. The carbon chain that may have a heteroatom corresponds to the binding portion between the oligonucleotide (that is, the recognition site) having a sequence complementary to the target RNA and the fluorescent coloring unit, so the starting point is , An atom bonded to the oxygen atom existing at the 5′-end of the recognition site (that is, the oxygen atom remaining after removing the hydrogen atom from the 5′-terminal hydroxyl group), and the end point is bonded to the fluorescent coloring unit. It becomes an atom.

  As the fluorescence coloring unit, a group that emits fluorescence upon irradiation with light such as ultraviolet light or visible light, and a group that quenches fluorescence by a PET phenomenon from a nitrogen atom provided in the vicinity thereof is used. Examples of such a group include a divalent organic group obtained by removing two substituents which may be hydrogen atoms from a condensed polycyclic aromatic compound which may have a hetero atom. The reason for being a “divalent organic group” is that the fluorescent coloring unit is bound to the divalent linker unit and also to the boronic acid unit described later.

  Examples of the condensed polycyclic aromatic compound which may have a hetero atom include anthracene, naphthacene, pentacene, benzopyrene, chrysene, pyrene, triphenylene, perylene, fluorescein and the like. Among these, anthracene and pyrene can be preferably exemplified.

  The boronic acid unit is a unit bonded to the above-described fluorescence coloring unit and is represented by the following general formula (1). As already explained, the boronic acid unit contains a phenylboronic acid group and a tertiary amino group. The phenylboronic acid group is used to form a bond with the cis-2,3-diol structure present at the 3'-end of the target RNA. And when light is irradiated to the reagent for RNA detection of the present invention, when the phenylboronic acid group is free, the fluorescence of the fluorescence coloring unit is quenched by PET of the unshared electron pair electron contained in the tertiary amino group, When phenylboronic acid forms a bond, the PET is not generated, and fluorescence from the fluorescence coloring unit is observed. Thus, the RNA detection reagent of the present invention can detect the presence or absence of target RNA in the solution.

In the general formula (1), R ¹ is a hydrogen atom or an alkyl group, and m and n are each independently an integer of 0 to 4. Further, the boronic acid unit represented by the general formula (1) is bound to the fluorescence coloring unit by a single bond with a wavy line. In the general formula (1), the boronic acid group may be bonded to any substitutable carbon atom in the benzene ring to which the boronic acid group is bonded. R ¹ is preferably exemplified by a methyl group.

  Although the reagent for RNA detection of this invention is equipped with the above structure, the compound represented by following General formula (2) can be mentioned as a preferable example of the reagent for RNA detection of this invention.

In the above general formula (2), Nu is a group obtained by removing the 5′-terminal hydroxyl group from an oligonucleotide having a sequence complementary to the target RNA. X is a divalent organic group obtained by removing two substituents which may be hydrogen atoms from a condensed polycyclic aromatic compound which may have a hetero atom, and is the above-described fluorescence coloring unit. L is the linker unit which is a divalent organic group. R ¹ is the same as in the general formula (1). In the above general formula (2), the oxygen atom present between Nu and L is the oxygen atom remaining after removing the hydrogen atom from the 5′-terminal hydroxyl group of the oligonucleotide. Details of each unit are as described above. As an example of the reagent for RNA detection which showed L (linker unit) in the said General formula (2) more specifically, the compound represented by following General formula (3) can be mentioned.

In the general formula (3), Nu is a group obtained by removing the 5′-terminal hydroxyl group from an oligonucleotide having a sequence complementary to the target RNA as described above. X is a divalent organic group obtained by removing two substituents, which may be hydrogen atoms, from the condensed polycyclic aromatic compound which may have a heteroatom, as described above, and is the fluorescence coloring unit. R ¹ is the same as in the general formula (1), R ² is a hydrogen atom or an alkyl group, Z is a divalent group or a single bond, and n is an integer of 1 to 18. In the general formula (3), the oxygen atom present between Nu and Z is an oxygen atom remaining after removing the hydrogen atom from the 5′-terminal hydroxyl group of the oligonucleotide.

In the above general formula (3), the portion corresponding to the linker unit is from Z to C (═O) N (R ² ) CH ₂ . This linker unit will comprise Z and methylene groups as linking groups. The carbon chain in the linker unit becomes an alkylene group and an amide group.

  As Z which is one bonding group in the linker unit, —P (═O) (— OH) O— can be preferably exemplified. In this case, the oligonucleotide represented by Nu and the other part composed of the linker unit, the fluorescence coloring unit and the boronic acid unit are linked by a phosphodiester bond (phosphodiester bond). For example, a phosphoramidide compound having a structure corresponding to Z to a boronic acid unit in the general formula (3) is prepared, and the phosphoramidide compound is used as a final step when an oligonucleotide represented by Nu is synthesized by an automatic synthesizer. If the process of making it act is performed, Z will correspond to a phosphodiester bond. Such a method is preferable because the RNA detection reagent of the present invention can be synthesized by an automated oligonucleotide synthesizer. Next, as an example of a method for synthesizing the reagent for detecting RNA of the present invention, a synthesis method using the phosphoramidide compound will be described.

  In this synthesis method, a phosphoramidide compound represented by the following general formula (4) is used as a reagent for synthesizing an RNA detection reagent. A reagent for synthesizing such a reagent for RNA detection is also one aspect of the present invention. The phosphoramidide compound represented by the following general formula (4) includes a portion corresponding to the above-described linker unit, fluorescent coloring unit and boronic acid unit having a protecting group, and acts on the 5′-end of the oligonucleotide. These units can be introduced into the oligonucleotide. That is, the RNA detection of the present invention is carried out by allowing the phosphoramidide compound represented by the following general formula (4) to act on the 5′-end of an oligonucleotide having a sequence complementary to the target RNA, followed by deprotection. Reagents are synthesized.

In the general formula (4), X is a divalent organic group obtained by removing two substituents which may be hydrogen atoms from the condensed polycyclic aromatic compound which may have a hetero atom. This X corresponds to the above-described fluorescence coloring unit. L is a divalent organic group and corresponds to the linker unit described above. R ¹ is a hydrogen atom or an alkyl group, and R ³ and R ⁴ are each independently a protecting group for boronic acid. R ³ and R ⁴ may be bonded to each other to form a ring. R ⁵ is a protecting group for a phosphite group, and R ⁶ and R ⁷ are each independently an alkyl group. R ⁶ and R ⁷ may be bonded to each other to form a ring.

Examples of R ³ and R ^{4 which} are boronic acid protecting groups include known boronic acid protecting groups without any particular limitation. As such a protecting group, N-methyliminodiacetic acid ester (also referred to as MIDA protecting group) can be preferably exemplified. The MIDA protecting group is introduced by allowing N-methyliminodiacetic acid to act on phenylboronic acid contained in the boronic acid unit. Moreover, since various boronic acid compounds having a MIDA protecting group are commercially available, the phosphoramidide compound of the above compound (4) may be synthesized using this. To assist in understanding the MIDA protecting group, the chemical formula of phenylboronic acid into which the MIDA protecting group has been introduced is shown below. As can be understood from the chemical formula below, when a MIDA protecting group is used as a protecting group for boronic acid, R ³ and R ⁴ in the general formula (4) are bonded to each other to form a ring.

  MIDA protecting groups are easily deprotected under basic conditions. For this reason, the RNA detection reagent of the present invention synthesized on a solid phase carrier with a protective group in the oligonucleotide moiety and phenylboronic acid group using an oligonucleotide automatic synthesizer is immobilized under basic conditions. When cutting out from the phase carrier, all of these protecting groups are eliminated, and the RNA detection reagent of the present invention is obtained.

R ⁵ which is a protecting group for the phosphite group is not particularly limited. Examples of such protecting groups include alkyl groups such as methyl group, ethyl group, and isopropyl group, and cyanoethyl group. R ⁶ and R ⁷ which are each independently an alkyl group are not particularly limited. An isopropyl group can be exemplified as such an alkyl group. R ⁶ and R ⁷ may be bonded to each other to form a ring.

  As an example of the phosphoramidide compound showing L (linker unit) in the general formula (4) more specifically, a compound represented by the following general formula (5) can be given.

In the general formula (5), X, R ¹ , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ are the same as those in the general formula (4). R ² is a hydrogen atom or an alkyl group, preferably a methyl group. n is an integer of 1-18.

  More specific examples of such phosphoramidide compounds include compounds represented by the following chemical formula (6).

  Next, an example of the synthesis route of the phosphoramidide compound is shown below. In the following, a synthesis route for the phosphoramidide compound represented by the above chemical formula (6) is shown as an example of a reagent for synthesizing the reagent for RNA detection of the present invention. However, the present invention is represented by the above chemical formula (6). It is not limited to the phosphoramidide compound represented.

In the above synthetic route, Boc represents a tert-butyloxycarbonyl group, Boc ₂ O represents di-tert-butyl dicarbonate, SO ₃ -pyridine represents a sulfur trioxide-pyridine complex, and TEA represents Represents triethylamine, MIDA represents N-methyliminodiacetic acid, and DIPEA represents N, N-diisopropylethylamine.

  The above phosphoramidide compound can be used as a reagent for synthesizing a reagent for RNA detection in an automated oligonucleotide synthesizer to which the phosphoamidide method is applied. The phosphoamidide method used in an oligonucleotide automatic synthesizer is a solid phase method, in which oligonucleotides are chemically synthesized on the surface of carrier particles, which are solid phases, using a condensation reaction of a phosphoamidide compound. As a procedure for synthesizing the RNA detection reagent of the present invention, first, an oligonucleotide having a sequence complementary to a target RNA is synthesized according to a known phosphoramidide method, and then the 5′-terminal of the oligonucleotide is synthesized. By reacting the above phosphoramidide compound with the hydroxyl group deprotected, a linker unit, a fluorescence coloring unit and a boronic acid unit are introduced at the 5′-end of the oligonucleotide, and finally, under basic conditions. Then, the oligonucleotide is cut out from the surface of the solid support. At the time of this excision, various protecting groups added to the oligonucleotide and protecting groups added to phenylboronic acid are eliminated, and the RNA detection reagent of the present invention is obtained. In short, since the reagent for synthesizing the reagent for RNA detection of the present invention has a phosphoamidide site in the same manner as the nucleoside phosphoamidide used for extending the oligonucleotide, it can be applied to an automatic synthesizer in the same manner as the nucleoside phosphoamidide. Is possible. Accordingly, by appropriately programming an automatic synthesizer, a reagent for synthesizing the RNA detection reagent of the present invention is automatically introduced into the reaction system after the oligonucleotide has reached the desired length, and the oligonucleotide 5 ' It is possible to introduce a linker unit, a fluorescence coloring unit and a boronic acid unit at the end on the-side.

  As described above, the RNA detection reagent of the present invention and the reagent for synthesizing the RNA detection reagent have been described. However, the RNA detection reagent of the present invention was added to the test object, and the test object was irradiated with ultraviolet rays or visible light. In this case, a test method for determining whether the target RNA is included in the test target by observing whether the fluorescence of the RNA detection reagent is enhanced is also one of the present inventions. It is. Since such an inspection method has already been described in the description of the reagent for RNA detection of the present invention, description thereof is omitted here.

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

Synthesis Example 1 Synthesis of trans-dispiro [oxirane-2,9 ′ (10H) -anthracene-10 ′, 2 ″ -oxirane] (8)

Commercially available anthraquinone (7) (6.2 g, 30 mmol) was dissolved in 120 mL of dry DMSO (dimethyl sulfoxide), and the system was purged with argon gas. Then, sodium hydride (2.2 eq, 2.4 g, 66 mmol, purity) 60%) was added, and the mixture was stirred at room temperature and protected from light, and trimethylsulfonium iodide (2.2 eq, 13.4 g, 66 mmol) dissolved in 80 mL of dry DMSO was added dropwise thereto over 30 minutes. After 5 hours, the progress of the reaction was confirmed by TLC (thin layer chromatography) and filtered through a glass filter. The filtrate was added to 300 mL of ice water and stirred for 1 hour. The precipitated solid was collected by filtration and washed with water to obtain the target compound (8) as a yellow solid (4.9 g, 20.6 mmol, yield 69%).
The physical property values of compound (8) are as follows.
¹ H NMR (500 MHz, DMSO-d ₆ ) δ (ppm): 7.42-7.40 (m, 4H, An-1, 4, 5, 8), 7.32-7.29 (m, 4H , An-2,3,6,7), 3.12 ( s, 4H, O-CH 2)

[Synthesis Example 2] Synthesis of 10-hydroxymethyl-9-anthraldehyde (9)

Compound (8) (4.9 g, 20.6 mmol) was dissolved in 400 mL of dry acetonitrile, lithium bromide (2.0 eq, 3.6 g, 41.2 mmol) was added, and the mixture was stirred at 60 ° C. After 91 hours, the progress of the reaction was confirmed by TLC, the reaction solution was cooled to room temperature, and further cooled at −40 ° C. for 30 minutes. The precipitated solid was collected by filtration and washed with water to obtain the target compound (9) as a yellow solid (3.3 g, 13.8 mmol, yield 67%).
The physical property values of compound (9) are as follows.
¹ H NMR (500 MHz, DMSO-d ₆ ) δ (ppm): 11.46 (s, 1H, CH═O), 8.95 (d, J = 9.0 Hz, 2H, An-1, 8), 8.61 (d, J = 8.5 Hz, 2H, An-4, 5), 7.74-7.65 (m, 4H, An-2, 3, 6, 7), 5.57 (t, J = 5.3 Hz, 1H, OH), 5.49 (d, J = 5.5 Hz, 2H, CH ₂ )

Synthesis Example 3 Synthesis of (10-methylaminomethylanthracen-9-yl) methanol (10)

To the compound (9) (3.3 g, 13.8 mmol) were added 150 mL of methanol, 70 mL of THF (tetrahydrofuran), and 30 mL of a 40% aqueous solution of methylamine, and the mixture was stirred at room temperature for 1 hour in an argon atmosphere. To the solution was added sodium borohydride (2.8 eq, 1.5 g, 38.6 mmol), and the mixture was further stirred for 4 hours, and the progress of the reaction was confirmed by TLC. The solvent was distilled off under reduced pressure, dissolved in ethyl acetate, washed with water, and then washed with saturated brine. The aqueous solution used for washing was also extracted with ethyl acetate. The organic layer was collected, dried over magnesium sulfate, filtered and the solvent was distilled off to obtain the target compound (10) as a yellow solid (3.3 g, 13.2 mmol, yield 95%). .
The physical property values of compound (10) are as follows.
¹ H NMR (500 MHz, DMSO-d ₆ ) δ (ppm): 8.49-8.43 (m, 4H, An-1, 4, 5, 8), 7.61-7.54 (m, 4H) , An-2, 3, 6, 7), 5.43 (d, J = 5.5 Hz, 2H, CH ₂ ), 5.30 (t, J = 5.3 Hz, 1H, OH), 4.59 _{(s, 2H, N-CH} 2)

Synthesis Example 4 Synthesis of (10-hydroxymethylanthracen-9-ylmethyl) methylcarbamic acid tert-butyl ester (11)
(In the above chemical formula, Boc represents a tert-butyloxycarbonyl group. The same applies hereinafter.)

Methanol 180 mL and di-tert-butyl dicarbonate (2.1 eq, 6.4 mL, 27.7 mmol) were added to compound (10) (3.3 g, 13.2 mmol), and the mixture was stirred at room temperature for 64 hours. The progress of the reaction was confirmed by TLC, the solvent was distilled off under reduced pressure, the residue was dissolved in ethyl acetate, washed twice with water, once with 10% aqueous sodium carbonate and twice with aqueous sodium chloride. The aqueous solution used for washing was also extracted with ethyl acetate. The organic layer was collected, dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The obtained residue was purified by silica gel column chromatography. Fractions containing the desired product were collected, and the solvent was distilled off under reduced pressure to obtain the desired compound (11) as a pale yellow foamy solid (4.0 g, 11.5 mmol, yield 87%).
The physical property values of compound (11) are as follows.
¹ H NMR (500 MHz, DMSO-d ₆ ) δ (ppm): 8.54-8.50 (m, 4H, An-1, 4, 5, 8), 7.59-7.58 (m, 4H) _{, An-2,3,6,7), 5.46-5.45 (} m, 4H, N-CH 2), 5.35 (m, 1H, OH), 2.38 (s, 3H, N _{-CH 3), 1.58-1.47 (br,} 9H, H-Boc)

Synthesis Example 5 Synthesis of (10-formylanthracen-9-ylmethyl) methylcarbamic acid tert-butyl ester (12)

Compound (11) (4.0 g, 11.5 mmol) is dissolved in 40 mL of dry DMSO, added with 40 mL of triethylamine (TEA), stirred at room temperature under an argon atmosphere, and sulfur trioxide-pyridine dissolved in 35 mL of dry DMSO. The complex (SO ₃ -pyridine, 7.0 eq, 12.6 g, 80.5 mmol) was added dropwise over 30 minutes. After 1 hour, the progress of the reaction was confirmed by TLC, and the reaction solution was poured into 100 mL of ice water. The reaction solution was washed into a separatory funnel, extracted with ethyl acetate, washed twice with water and twice with a 10% aqueous sodium hypochlorite solution. The aqueous solution used for washing was also extracted with ethyl acetate. The organic layer was collected, dried over magnesium sulfate, filtered, and the solvent was distilled off under reduced pressure to quantitatively obtain the target compound (12) as a yellow candy-like solid (5.1 g, 14.6 mmol). I got it.
The physical property values of compound (12) are as follows.
¹ H NMR (500 MHz, DMSO-d ₆ ) δ (ppm): 11.46 (s, 1H, CH═O), 8.92 (d, J = 9.0 Hz, 2H, An-4, 5), 8.63 (d, J = 9.0 Hz, 2H, An-1, 8), 7.74-7.61 (m, 4H, An-2, 3, 6, 7), 5.54 (s, _{2H, N-CH 2),} 2.40 (s, 3H, N-CH 3), 1.75-1.48 (brs, 9H, H-Boc)

Synthesis Example 6 Synthesis of methyl- (10-methylaminomethylanthracen-9-ylmethyl) carbamic acid tert-butyl ester (13)

To compound (12) (5.1 g, 14.6 mmol) were added methanol 120 mL, THF 120 mL, and methylamine 40 mL aqueous solution 60 mL, and the mixture was stirred at room temperature for 30 minutes in an argon atmosphere. To the solution was added sodium borohydride (4.0 eq, 2.2 g, 54.4 mmol) and stirred for another 2 hours. The progress of the reaction was confirmed by TLC, and the solvent was distilled off under reduced pressure. The residue was dissolved in ethyl acetate and washed 3 times with water. The aqueous solution used for washing was also extracted with ethyl acetate. The organic layer was collected, dried over magnesium sulfate, filtered and the solvent was distilled off to obtain the target compound (13) as a yellow solid (4.0 g, 11.1 mmol, yield 76%). .
The physical property values of compound (13) are as follows.
¹ H NMR (500 MHz, DMSO-d ₆ ) δ (ppm): 8.54-8.49 (m, 4H, An-1, 4, 5, 8), 7.61-7.55 (m, 4H) _{, An-2,3,6,7), 5.46 (} s, 2H, N-CH 2), 4.56 (s, 2H, N-CH 2), 2.37 (s, 3H, N- _{CH 3), 1.47 (br,} 9H, H-Boc)

Synthesis Example 7 Synthesis of tert-butyl ((10-((6-hydroxy-N-methylhexaneamido) methyl) anthracen-9-yl) methyl) (methyl) carbamate (14)

Compound (13) (2.0 g, 5.0 mmol) was dissolved by adding 30 mL of methanol, and 6-hydroxycaproic acid (1.2 eq, 786 mg, 6.0 mmol) and 4- (4,6-dimethoxy-1, 3,5-Triazin-2-yl) -4-methylmorpholinium (3.0 eq, 4.2 g, 15 mmol) was added, the inside of the system was replaced with argon, and the mixture was stirred at room temperature for 45 minutes. The progress of the reaction was confirmed by TLC, and the solvent was distilled off under reduced pressure. The residue was dissolved in ethyl acetate and washed twice with water and twice with aqueous sodium chloride solution. The aqueous solution used for washing was also extracted with ethyl acetate. The organic layer was collected, dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The obtained residue was purified by silica gel column chromatography. Fractions containing the desired product were collected, and the solvent was distilled off under reduced pressure to obtain the desired compound (14) as a yellow foamy solid (1.9 g, 3.97 mmol, yield 79%).
The physical property values of compound (14) are as follows.
¹ H NMR (600 MHz, DMSO-d ₆ ) δ (ppm): 8.52 (m, 4H, An-1, 4, 5, 8), 7.54 (m, 4H, An-2, 3, 6 7), 5.59 (s, 2H, N—CH ₂ ), 5.45 (s, 2H, N—CH ₂ ), 4.31 (t, J = 5.2 Hz, 1H, OH), 3 .35 (CH ₂ bound to m, 2H, OH), 2.49 (s, 3H, N—CH ₃ ), 2.35 (s, 3H, N—CH ₃ ), 2.29 (t, J _{= 7.2Hz, 2H, O = C} -CH 2), 1.46 (br, 15H, H-Boc, CH 2 -CH 2 -CH 2)

Synthesis Example 8 Synthesis of 6-hydroxy-N-methyl-N-((10-((methylamino) methyl) anthracen-9-yl) methyl) hexanamide (15)

Compound (14) (1.9 g, 3.97 mmol) was dissolved in 80 mL of dichloromethane, 30 mL of trifluoroacetic acid was added, the inside of the system was purged with argon, and the mixture was stirred at room temperature for 1 hour. The progress of the reaction was confirmed by TLC, and the solvent was distilled off under reduced pressure. The residue was dissolved in chloroform and washed once with water and twice with an aqueous solution of sodium chloride and sodium carbonate. Extraction was also performed with chloroform from the aqueous solution used for washing. The organic layer was collected, dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The obtained residue was purified by silica gel column chromatography. Fractions containing the desired product were collected, and the solvent was distilled off under reduced pressure to obtain the desired compound (15) as a yellow candy-like solid (1.1 g, 2.88 mmol, yield 73%).
The physical property values of compound (15) are as follows.
¹ H NMR (500 MHz, DMSO-d ₆ ) δ (ppm): 8.44 (m, 4H, An-1, 4, 5, 8), 7.56 (m, 4H, An-2, 3, 6 7), 5.57 (s, 2H, N—CH ₂ ), 4.77 (s, 2H, N—CH ₂ ), 4.30 (s, 1H, NH), 3.33 (m, 2H) , CH ₂ bonded to OH), 2.58 (s, 3H, N—CH ₃ ), 2.45 (CH ₃ bonded to s, 3H, NH), 2.29 (t, J = 7.2 Hz). _{, 2H, O = C-CH} 2), 1.54-1.32 (m, 6H, CH 2 -CH 2 -CH 2)

[Synthesis Example 9] Synthesis of Compound (16)

Compound (15) (1.1 g, 2.99 mmol) is dissolved in 25 mL of DMF (dimethylformamide), 4-formylphenylboronic acid-N-methyliminodiacetic ester (1.2 eq, 937 mg, 3.59 mmol) and sodium Triacetoxyborohydride (1.4 eq, 889 mg, 4.19 mmol) was added, and the system was purged with argon, followed by stirring at room temperature for 23 hours. After confirming the progress of the reaction by TLC, ethyl acetate was added to the reaction solution for extraction, and the organic layer was washed twice with water and three times with an aqueous sodium chloride solution. The aqueous solution used for washing was extracted 10 times with ethyl acetate. The organic layer was collected, dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The obtained residue was purified by silica gel column chromatography. Fractions containing the target product are collected, the solvent is distilled off under reduced pressure, dissolved in a small amount of dichloromethane, the target product and by-products are separated by reprecipitation, and the filtrate is concentrated under reduced pressure. A certain compound (16) was obtained as a pale yellow foamy solid (1.1 g, 1.73 mmol, yield 58%).
The physical property values of compound (16) are as follows.
¹ H NMR (500 MHz, DMSO-d ₆ ) δ (ppm): 8.54 (m, 2H, An-1, 8) 8.40 (d, 2H, An-4, 5), 7.52 (m , 4H, phenyl), 7.33 (d, J = 8.0 Hz, 2H, An-2, 7), 7.24 (d, J = 8.0 Hz, 2H, An-3, 6), 5. 56 (s, 2H, N—CH ₃ ), 4.47 (s, 2H, N—CH ₃ ), 4.32 (t, J = 5.0 Hz, 1H, OH), 4.25 (d, J = 17.0 Hz, 2H, N—CH ₂ —C═O), 4.02 (d, J = 17.0 Hz, 2 H, N—CH ₂ —C═O), 3.65 (s, 2H, N) _{-CH 2), 3.34 (q,} J = 6.3Hz, 2H, CH 2) bonded to _{OH, 2.49 (s, 3H,} N-CH 3), 2.40 (s, 3H, N -CH _{), 2.28 (t, J =} 7.3Hz, 2H, O = C-CH 2), 2.04 (s, 3H, N-CH 3), 1.53-1.28 (m, 6H, CH ₂ -CH ₂ -CH ₂₎

[Synthesis Example 10] Synthesis of phosphoramidide compound (6)

Compound (16) (400 mg, 0.64 mmol) was azeotropically dehydrated with dry pyridine, azeotropically removed with dry toluene, the interior of the system was purged with argon, dissolved in dichloromethane, and N, N-diisopropylethylamine ( DIPEA) (2.4 eq, 268 μL, 1.54 mmol) and 2-cyanoethyl-N, N-diisopropylchlorophosphoamidide (1.2 eq, 191 μL, 0.77 mmol) were added and stirred at room temperature for 1 hour under an argon atmosphere. did. After confirming the progress of the reaction by TLC, the solvent was distilled off under reduced pressure. The residue was dissolved in chloroform and washed twice with an aqueous sodium bicarbonate solution and twice with an aqueous sodium chloride solution. The organic layer was collected, dried over magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The residue was dissolved in acetonitrile and purified by recycle preparative HPLC. Fractions containing the desired product were collected, and the solvent was distilled off under reduced pressure to obtain the desired compound (6) as a pale yellow foamy solid (246 mg, 0.30 mmol, 47% yield).
The physical property values of compound (6) are as follows.
¹ H NMR (600 MHz, CDCl ₃ ) δ (ppm): 8.54 (m, 2H, An-1, 8), 8.33 (m, 2H, An-4, 5), 7.53 (m, 4H, phenyl), 7.41 (d, J = 7.8 Hz, An-2, 3), 7.32 (d, J = 8.4 Hz, 2H, An-6, 7), 5.66 (s) , 2H, N—CH ₂ ), 4.51 (s, 2H, N—CH ₂ ), 3.95 (d, J = 16.2 Hz, 2H, N—CH ₂ —C═O), 3.85. (M, 2H, O—CH ₂ ), 3.77 (m, 2H, O—CH ₂ ), 3.72 (d, J = 16.8 Hz, 2H, N—CH ₂ —C═O), 3 .59 (m, 2H, N—CH), 2.62 (t, J = 6.3 Hz, 2H, CH ₂ —CN), 2.56 (s, 3H, N—CH ₃ ), 2.47 ( s, 3H, N _{-CH 3), 2.37 (t,} J = 7.5Hz, 2H, CH 2 -CH 2 -C = O), 2.24 (s, 3H, N-CH 3), 1.74-1. _{27 (m, 6H, CH 2} -CH 2 -CH 2), 1.18 (m, 12H, i-Pr)
³¹ P NMR (243 MHz, CDCl ₃ ) δ (ppm): 147.8

[Example 1] Synthesis of oligonucleotides Using an oligonucleotide automatic synthesizer, three types of oligonucleotides having sequences represented by the following (B12), (T12) and (U12) were synthesized. What is indicated by the symbol “B” in (B12) is a linker unit, a fluorescence coloring unit and a boronic acid unit which are added to the 5′-end of the oligonucleotide and represented by the following chemical formula. This was added to the 5′-end of the oligonucleotide by allowing compound (6) to act after the synthesis of the oligonucleotide was completed by an automatic synthesizer. In view of the fact that RNA is relatively unstable in solution, in the following (B12), (T12), and (U12), oligonucleotides derived from nucleotides in DNA were used. However, only (U12), the 3′-terminal nucleotide was uridine (U) so that the 3′-terminal had the same structure as RNA, and this was used as a simulated oligonucleotide for RNA. (B12) and (T12) are oligonucleotides having base sequences complementary to each other, and (B12) and (U12) are oligonucleotides having base sequences complementary to each other. That is, (B12) is a book comprising an oligonucleotide having a sequence complementary to the target RNA (U12, which is an RNA mimetic oligonucleotide), a divalent linker unit, a fluorescence coloring unit, and a boronic acid unit. It is a reagent for RNA detection of the invention.

5 'B AAC GCA TGT CAC 3' (B12)
5 'GTG ACA TGC GTT 3' (T12)
5 'GTG ACA TGC GTU 3' (U12)

(In the above chemical formula, the wavy line represents the oligonucleotide chain bound to “B”.)

In addition, in the automatic synthesis | combination of oligonucleotide, the solid-phase synthesis method according to the phosphoamidide method was used on the automatic synthesizer (Applied Biosystems 394 DNA / RNA Synthesizer). Commercially available products from Glen Research were used as various phosphoramidite units for synthesizing oligonucleotides. A protocol starting from a nucleoside protector (1 μmol) bound to a solid phase carrier (CPG) and using a protocol modified by Applied Biosystems with some modifications was used. That is, the condensation time of the amidite unit of compound (6) was set to 300 seconds, and the condensation time was longer than that of a commonly used natural amidite unit. For (T12) and (U12), the synthesis was completed with the DMTr group (dimethoxytrityl protecting group) left at the 5′-end. At the end of the synthesis, argon gas was passed for 100 seconds to dry the solid support. The solid support to which the fully protected oligonucleotide was bound was transferred to a vial, and the oligonucleotide was cut out from the solid support. By this excision operation, the synthesized oligonucleotide is excised from the solid phase carrier and all the protecting groups bonded to the oligonucleotide are eliminated. For the cutting operation, 28% ammonia aqueous solution was used for (T12) and (U12), 0.4M sodium hydroxide methanol solution was used for (B12), the solid phase carrier was removed by cotton plug filtration, and the purification operation was performed. went. Then, the target object was fractionated by HPLC using a reverse phase silica gel carrier. Each obtained oligonucleotide was analyzed by UV measurement and MALDI-TOF MASS, and confirmed to be the target product. The chemical reaction formula in the cutting-out operation from the solid phase carrier (B12) is shown below. In the following chemical reaction formula, those indicated by black circles represent a solid phase carrier, Base represents a nucleobase, and B ^pro represents a nucleobase having a protecting group.

[Fluorescence measurement test]
For each of the oligonucleotide chain having a diol structure at the end (U12) and the diol structure not existing at the end of the oligonucleotide chain (T12), (B12) having an oligonucleotide chain complementary thereto was added. The fluorescence spectrum was measured. This measurement was performed with phosphate buffer solutions having pHs of (a) 7.0, (b) 7.5, and (c) 8.0, respectively. The measurement results are shown in FIG. In addition, in FIG. 1, the fluorescence spectrum about each of (B12) + (U12), (B12) + (T12), and (B12) independent as a control | contrast is shown. The fluorescence spectrum was measured in a 100 mM phosphate buffer solution containing 1 M NaClO ₄ with a wavelength of excitation light of 379 nm and concentrations of (U12), (T12) and (B12) of 2 μM. .

  As shown in FIG. 1, the fluorescence spectrum in (B12) + (T12) was almost the same as the fluorescence spectrum in the case of (B12) alone with no complementary strand added. That is, when (T12) is used as a complementary strand, no boronic acid ester is formed, so that it is considered that PET is not eliminated and the fluorescence intensity is not changed. On the other hand, the intensity of the fluorescence spectrum in (U12) + (B12) increased compared to the case of (B12) alone or (T12) + (B12). This is because (B12), which is the reagent for detecting RNA of the present invention, recognizes the cis-2,3-diol structure at the 3′-end of (U12) and forms a boronic ester, thereby eliminating PET. It is thought that this is caused by the recovery of fluorescence. That is, according to the reagent for RNA detection of the present invention, it is understood that whether or not target RNA is present in a solution can be determined by observation of fluorescence intensity.

Claims (8)

Oligonucleotide having a sequence complementary to the target RNA, a divalent linker unit condensed at one end with the hydroxyl group at the 5′-end of the oligonucleotide, and fluorescence bound to the other end of the divalent linker unit A reagent for RNA detection comprising a color developing unit and a boronic acid unit represented by the following general formula (1) bonded to the fluorescent color developing unit.
(In the general formula (1), R ¹ is a hydrogen atom or an alkyl group, and m and n are each independently an integer of 0 to 4. The boronic acid unit represented by the general formula (1) Is bound to the fluorescent coloring unit by a single bond with a wavy line.)   The reagent for RNA detection according to claim 1, wherein the fluorescent coloring unit is a divalent organic group obtained by removing two substituents which may be hydrogen atoms from a condensed polycyclic aromatic compound which may have a hetero atom. . The reagent for RNA detection of Claim 1 or 2 represented by following General formula (2).
(In the general formula (2), Nu is a group formed by removing the 5′-terminal hydroxyl group from the oligonucleotide, and X may be a hydrogen atom from a condensed polycyclic aromatic compound which may have a hetero atom. The fluorescent coloring unit that is a divalent organic group obtained by removing two substituents, L is the linker unit that is a divalent organic group, and R ¹ is the same as in the general formula (1). In the above general formula (2), the oxygen atom present between Nu and L means the oxygen atom remaining after removing the hydrogen atom from the 5′-terminal hydroxyl group of the oligonucleotide. ) The reagent for RNA detection of any one of Claims 1-3 represented by following General formula (3).
(In the above general formula (3), Nu is a group formed by removing the 5′-terminal hydroxyl group from the oligonucleotide, and X may be a hydrogen atom from a condensed polycyclic aromatic compound which may have a hetero atom. In the fluorescent color developing unit, which is a divalent organic group obtained by removing two substituents, R ¹ is the same as in the general formula (1), R ² is a hydrogen atom or an alkyl group, and Z is It is a divalent group or a single bond, and n is an integer of 1 to 18. In the general formula (3), the oxygen atom present between Nu and Z is the 5′- of the oligonucleotide. (This means the oxygen atom remaining after removing the hydrogen atom from the terminal hydroxyl group.)   The reagent for RNA detection according to claim 4, wherein Z in the general formula (3) is -P (= O) (-OH) O-.   Whether or not the fluorescence of the RNA detection reagent is enhanced when the RNA detection reagent according to any one of claims 1 to 5 is added to the test object and the test object is irradiated with ultraviolet rays or visible light. A test method for determining whether or not a target RNA is contained in the test object by observing the above. A reagent for synthesizing a reagent for RNA detection represented by the following general formula (4).
(In the general formula (4), X is a divalent organic group obtained by removing two substituents which may be hydrogen atoms from a condensed polycyclic aromatic compound which may have a hetero atom, and L is 2 R ¹ is a hydrogen atom or an alkyl group, R ³ and R ⁴ are each independently a boronic acid protecting group, and R ³ and R ⁴ are bonded to each other to form a ring. R ⁵ is a protecting group for the phosphite group, R ⁶ and R ⁷ are each independently an alkyl group, and R ⁶ and R ⁷ may be bonded to each other to form a ring. ) The reagent of Claim 7 represented by following General formula (5).
(In the general formula (5), X, R ¹ , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ are the same as those in the general formula (4), and R ² is a hydrogen atom or an alkyl group. , N is an integer of 1-18.)