JPH05507619A - RNA hydrolysis/cleavage - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] RNA hydrolysis/cleavage field of invention The present invention relates to sequence-directed RNA hydrolysis under physiologically suitable conditions; In detail, oligodeoxynucleotides as RNA hydrolyzing agents dominated by 5 sequences. Covalently bound metal complexes and RNA cleavage in general, and in particular sequence-dominated RN In recent years, the hydrolysis of phosphate esters has been reported in the art because the chemistry is relevant to biological systems. Specifically, transition metal complexes have been studied in detail as Phosphate esters to indicate reactions catalyzed by enzymes of the sphatase type It has been investigated as a hydrolysis catalyst, and in detail it has been This is because it relates to the manipulation of the phosphodiester main chain of ). These reported studies is generally an activated p-nitrophenyl phosphate ester or phosphate. Phate anhydride (ATP) was used as a substrate (R, D, Cor nelius, Inorg.

Chem, 1980. 19. 1286-1290: P, R, Nartn an et al., J. Am.

Chem, Sac, 1982.104.2356-2361 and F.

Tafesse et al., [norg, Chem, 1985.24.2593- 2594).

Tetramine complex of Co(III) is adenosine 3',5'-monophosphate (cA MP) (J, Chin et al., Can J, Chem.

1987, 65.1882-1884) and adenosine-phosphate (AMP) (J. Chin et al., J. Am. Chem. Sac, 1989. It has been reported that the hydrolysis of 111゜4103-4105) can be promoted. has been done. Additionally, many divalent cations can catalyze RNA hydrolysis. It is also known that it can be done (J. J. Butzaw et al., Biochemist. y, 297L 10. 201B-2027 and J, J. Butzow et al., N. ture, 1975.254.358-359). Additionally, imidazole buffer Zinc ions in the presence of RNA dimer 3',5'-UpU at 80°C (R, Breslow et al., Proc. Natl, Acad, Sci, 1989.86.1746-1750 　　　　, ribonuclease enzymes are used to convert RNA in vivo and in vitro. It is known that the enzyme (The E zymes), Academic Press, New York, 1982, No. 15 Vol. 12, pp. 317-433), the active site of many glue bonucleases is (Richards et al., supra). , 1971, Vol. 4, Chapter 24, pp. 647-806). by ribonuclease In order to demonstrate the reaction catalyzed by This has been done using imidazole and imidazole derivatives as models. child In these reported studies, activated p-nitrogen instead of the RNA itself Phenyl phosphate esters have been commonly used as hydrolysis substrates ( Anslyn et al., J. Am. Chem. Soc., 1989° 111. 5972-5973 and Anslyn et al., supra, 8931-8932).

These activated esters mean that they are more easily hydrolyzed and also produce The p-nitropherulate anion, which is a substance, has a strong characteristic color, so the reaction is The truth is that it can be observed using simple spectrochemical techniques. are more easily studied than biological substrates. These analogs are biological substrates Although convenient models, they are not accurate models (Menger.

F, M, and Ladika, M, J, Am, Chem, Soc. ,, 1987.109°3145).

C, A, 5tejn et al., Cancer Research May, 1988. 48. 2659-2668 is an antiserum as a modulator of gene expression. The application of oligodeoxynucleotides is explained in detail, and more subtle and Proposing an effective method for producing limited catalytic hydrolysis of RNA by It was concluded that chemical groups can be attached to oligomers that can be used. 5te In et al., a suitable RNA hydrolyzing group is an imidazole group, which also known to be involved in phosphodiester hydrolysis in the active site of the enzyme It is chanting that it is true.

Hydrolysis of RNA with imidazole buffer has been reported in the art. (Breslow et al., J. Am. Chem.

Soc, 1986.108.2655-2659), the mechanism of this reaction has been studied. have been investigated (Anslyn et al., J. Am. Chem. Soc., 19 89. 111, 4473-4482). Based on these studies, the bifunctional monomer A general acid-general base mechanism has been proposed for RNA hydrolysis by imidazole. It was.

PCT International Specification published as WO38104300 on June 16, 1988 University patents, Inc. Patents, Inc.), as an endoribonuclease, RNA fragments with sequence specificity of cleavage greater than that of known polymerases. catalyzes the cleavage of a DNA restriction endonuclease and thus approaches that of a DNA restriction endonuclease. An RNA sequence-specific endonuclease that acts as a ribonuclease. An RNA enzyme or ribozyme is disclosed. Riboteam is , consisting entirely or partially of RNA itself, and therefore of Applicants' Extremely unstable chemically and enzymatically to DNA-based compounds . Such instability impairs the practical applicability of RNA hydrolyzing agents. . Furthermore, ribozyme has currently been developed using molecular biological methods with extremely low yield. Due to limited production capacity, it can only be obtained at a high price.

C, B. Chen et al., J. Am. Chem, Soc, 1988.　 110. 6570-6572 Niha, 1.10-7E+Throline-Copper ([r ) is effective for targeted cleavage of RNA and DNA. It has been described that it has an effect on chemical cleavage. This teaching does not apply to applicants' invention. As opposed to hydrolytic cleavage of RNA under physiologically suitable conditions, Concerning the oxidative cleavage of RNA by metal complexes bound to DNA at temperature It is. The amount of auxiliary reagents required to cause the oxidative degradation of Chen et al. Furthermore, the 1,10-phenanthroline copper-oligodeoxylate used The sinucleotide conjugate is itself oxidatively degraded under conditions of oxidative RNA cleavage. (The rate of oxidative cleavage by 1°10-phenanthroline steel thread is (The same applies to DNA).

P, C, 5chultz and collaborators have published a series of reports (D, R.

Corey et al., J. Am. Chem, Soc., 1988. 110. f614-1615; R, Zuckerman et al., J. Am, Chem. Soc, 1988. 110. 6592-6594, and R, Zuck erman et al., Proc. Natl. Acad.

Sci., USA, 1989.86.1766-1770), Enzymes co-bound to oligonucleotides (staphyloclonal nuclease, ribonuclease) site-selective NAs consisting of clease S or mutants of these parent enzymes) and the preparation of RNA hydrolyzing agents. One report (D, R.

Corey et al., Biochemistry, 1989.28.8277-82 86), when the position and length of the ring arm were changed, the touch Changes occurred in medium efficiency and site of cleavage. These nucleic acid hydrolyzing agents include several heavy It differs from the present invention in important aspects, in that the nucleic acid cleavage behavior is provided by an enzyme. , to the synthetic small molecule hydrolyzing agents (or enzyme mimics) disclosed by Applicants. therefore not provided; the enzyme is susceptible to proteolytic degradation by other enzymes; Staphylococcal nuclease depends on added calcium for its activity. Existence: Ribonuclease S is derived from ribonuclease A and S-protein. It is a non-covalent complex consisting of S-peptide and S-peptide. Loss of rate and specificity: oligonucleotide-staphylococcal nuclei The enzyme conjugate cleaves DNA as well as RNA and therefore Since it lacks specificity for only RNA and the results of nonspecific cleavage are common, This high activity is due to the uniqueness of the enzyme-based system developed by Schultz. specificity of enzyme-based systems is limited to physiologically favorable values (i.e., 0 increased artificially by lowering the temperature below

Considerable technology has been developed for the cleavage of RNA using enzymes and ribozymes. It's here. Currently, this technology has several methods for obtaining sequence-directed hydrolysis of RNA. at a physiologically suitable pH and as a synthetic analogue to the enzyme or lipozyme. There is no teaching of using metal complexes that hydrolytically cleave RNA at temperature. array-dominated RNA hydrolysis and RNA cleavage are ways to suppress the expression of specific idiotypes. today to prepare catalytic antisense oligonucleotides that act as a step. Highly desirable. From some practical points of view, as described by 5chultz et al. The brittle high molecular weight enzyme-oligonucleotide conjugates of the present invention are This is extremely disadvantageous compared to standard synthetic ribonucleases. On these practical points The ability to prepare more than trace amounts, adversely active or unknown and inactive Ability to prepare highly pure substances free of impurities; able to survive in vivo conditions and Ability to evade immunological responses and non-specific hydrolysis of DNA and RNA Has the ability to evade. This technique involves catalytically cleaving RNA to determine its sequence. nucleosides, as enzymes or synthetic analogs of liposomes to obtain molecular cleavage; using imidazole groups attached to nucleotides and oligodeoxynucleotides. There is no teaching that exists. Such RNA hydrolysis and cleavage is accomplished by catalytic antisene It is necessary to provide the basis for the development of new drugs.

Description of the invention The present invention relates to hydrolytic cleavage of RNA under physiologically suitable conditions.

The basis underlying the present invention is the combination of enzymes or ribozymes in the hydrolysis of RNA. The use of metal complexes that act as synthetic analogs. The present invention can be carried out via a suitable linker. at least one imidazole linker linked through two or more imidazole linkers; RNA production by a combination of two oligodeoxynucleotides containing a midazole group cleavage and nucleation via a suitable linker in the presence of an imidazole group in solution. Imidazo attached to a leoside, nucleotide or oligodeoxynucleotide It also relates to the cleavage of RNA by group. The imidazole group plays a role in the cleavage of RNA. It acts as a synthetic analog of the active site of an enzyme or ribozyme. For use herein Conjugates are metal complexes covalently bonded to nucleosides or nucleotides. a compound comprising two or more nucleosides or nucleotides covalently linked A compound comprising an imidazole group, or one or more compounds each co-bonded with an imidazole group. Refers to a combination of nucleosides or nucleotides in which an imidazole group is present.

As used herein, oligodeoxynucleotide conjugates refer to oligodeoxynucleotide conjugates. Oligodeoxynucleotide, a compound containing a metal complex co-bound to a nucleotide one or more imidazo co-conjugated to a leotide or oligodeoxynucleotide a compound comprising a metal complex co-bonded to a compound comprising a metal group, or two or more oligodeoxynucleotides, each having one or more covalently bonded imidazole groups; means a combination of oxynucleotides. Imidazole group used herein The term imidazole and nitrogen which retains the essential properties of imidazole Lewis (Cotton and Wilkinson, new Advanced Inorganic Chemistry, 1 988, Wiley, New York, 36 pages); Urc hin et al., (The Vocabulary of Organic Chemistry) NickChefflistry), Wiley, 1980, 248) or Brønsted (Orchin et al., Terminology of Organic Chemistry (TheV ocabulary of Organic Chemistry), Willi (Wiley), 1980, p. 249). or both.

A first aspect of the invention provides for the discovery of metal complexes useful for promoting RNA hydrolysis. related to A second aspect of the invention provides that the nucleoside or nucleotide The present invention relates to an RNA hydrolytically active conjugate comprising a bound metal complex. Main departure A third aspect of the invention is based on a metal complex covalently bonded to an oligodeoxynucleotide. Concerning the sequence-directed hydrolytic cleavage of RNA. Another aspect of the invention is that RN Nucleosides, nucleotides and oligodeoxynucleotides that act to promote the cleavage of A. Concerning finding two or more imidazole groups covalently bonded to nucleotides Ru. This oligodeoxynucleotide provides target recognition.

Therefore, the main objective of the present invention is to perform hydrolysis of RNA under physiologically suitable conditions. and to prepare for RNA cleavage. Other objects of the present invention include (1) RNA (2) to find a metal complex that is active in the hydrolysis of molecules that promote RNA cleavage to a greater extent than monoimidazole species2 (3) finding a molecule containing more than one imidazole group; (3) preserving RNA cleavage behavior; (4) preparation of conjugates with physiologically suitable conditions and RNA sequence control; Preparation of oligodeoxynucleotide conjugates effective for sequence-directed hydrolysis of RNA There is a product. Other objects and benefits of the invention may be found upon further consideration of this disclosure and claims. The structure of the compound below with bold numbers in parentheses is revealed by The corresponding "process diagram" is shown in 1-9.

Figure 1 shows RNA [poly(A)] prepared by the metal complex Zn-N-Me (CR). +1−+m] shows a typical example of HPLC analysis by applicants of the hydrolysis of . A, time = 0 hours: B, time 218 hours.

Figure 2 shows 3'-[4-[4'-methyl (2,2'-bimethyl) using CuCl2. pyridine)-4-ylcobutyl-phosphe-)]-]2'-deoxythymidine 3'-[4-[4'-methyl(2,2'- bipyridine)-4-ylcobutyl-phosphe i-2'-deoxy-thymidinea This figure shows the formation of the ammonium base (m(6)). represents the formation of metal complex nucleotide conjugates according to the present invention. . Figure 3 shows that compound (6) RNA [poly(A) l! -+s] hydrolytic cleavage is shown. this The figure depicts Applicants' Example V, in which a nucleic acid capable of hydrolyzing RNA The metal complex attached to the 3' position of leotide can hydrolyze RNA. It shows. A, control reaction, time = 48 hours; B, RNA from (6) reaction, time = 48 hours.

Figure 4 shows 5'-[4-[4'methyl(2,2'-bipyridine)-4-yl] rucobutyl-phosphe)]-]2'-deoxythymidine triethylammonium Hydrolytic opening of RNA [poly(A) + * - + * A fissure is shown. This figure represents Applicants' example vur, nucleotide 5 This shows that metal complexes attached to the ′ position can hydrolyze RNA. .

A, Control reaction, time = 48 hours, B, Reaction of (12) and RNA, time =48 hours.

Figure 5 shows 5-[3-[[2-[[4-[4'-methyl[2,2'-bibily] Zinc-4-yl]-1-oxbutyl]amino]ethylcoamino]-3-ox Sobrovir]-2'-deoxy-uridine copper (ID(17)) A fissure is shown. This figure represents Applicants' Example It has been shown that partially bound metal complexes can hydrolyze RNA.

A, control reaction, time = 48 hours; B, reaction between (17) and RNA, Time = 48 hours.

Figure 6 shows the Cu(bpy)” complex and DNA [poly(dA)l-1@] and RNA [poly(A)+t−+sco]. This illustration is from the book Applicants' example rt shows the observation of RNA Cu(bpy) complexes. This indicates that the cleavage is hydrolytic and not oxidative. ASDN Control reaction with A, time 218 hours, B, Cu (bpy)'' and DNA. reaction, time 218 hours; control reaction with C, RNA, time 218 hours; D, Reaction between Cu (bpy)'' and RNA, time 218 hours.

Figure 7 shows the sequence-directed hydrolysis of tRNA'' oligodeoxynucleotides. -Cu (bpy)” conjugate for polyacrylamide gel electrophoresis analysis Cytometry results are shown. This figure shows 1 under the conditions described in Example (32) and tRNA” after 7 h and polyacrylic acid of both reactions and control reactions. Figure 2 depicts Applicants' Example Xv showing a densitometric scan of a lylamide gel. Ru. A1 cleavage reaction: 1.29 PM tRNA tRNA, 6.4 M (31), 12.9 MMcu (trpy)”, 227MMCu (SO4), 50mM NaCl and 50mM) Lys buffer, pH=7.8; control reaction: 1.29Mt RNA'', 227MCu (SO4), 50mM NaCl and 50mM Lys buffer pH = 7.8; B, cleavage reaction: 1,29PM tRNA'', 6.4 M(31), 12.9MMCu (trpy)", 227MMCu (SO4 ), 50mM NaCl and 50mM) Lys buffer pH = 7.8; control Le reaction = 1.2 μM tRNA”’, 12.9 MMCu (trpy)”, 22 7MMcu(SO4) and 6.4μM 14v--oligodeoxynucleotide 5' -Ho- TGACGGCAGATTTA-OH-3'.

Figure 8 shows the cleavage of 32P-labeled RNA with compound (5A) and the cleavage of 32P-labeled RNA with compound (5A). An autoradiograph is shown showing that 6A) is ineffective in RNA cleavage. ing.

Lane #1. RNA control t=2 hours: lane #2.1=0 hours; lane #3, t=0.5 hours: Lane #4, t=1 hour; Lane #5, t=1.5 hours : Lane #6, t=2.0 hours, Lanes #7 and #8.2mM (6A) and RN Reaction with A: lane #7.1 = 0 hours; lane #8, t = 2 hours.

Figure 10 shows two oligodeoxynucleotides juxtaposed in a manner that enhances RNA cleavage. Simulates the use of leotide conjugates, labeled antisense probe 1 and probe 2. A formal representation is shown.

Process Scheme 1 depicts the synthesis of compound (5) as described in Applicants' Example III .

Process scheme 2 depicts the synthesis of compound (11) described in Applicants' Example Vl. .

Process Scheme 3 depicts the synthesis of compound (16) described in Applicants' Example IX .

Process scheme 4 shows the combination of compounds (20) and (23) described in Applicants' Example Xll. It represents growth.

Process Scheme 5 depicts the synthesis of compound (30) described in Applicants' Example XIV. Ru.

Process diagram 6 shows the production of tRNA'' by compound (32) described in Applicants' Example Xv. Represents sequence-dominated cleavage.

Process Scheme 7 depicts the synthesis of compound (5A) described in Applicants' Example XVI. Ru.

Step 8 shows the arrangement of RNA with compound (5A) described in Applicants' Example XVII. It represents the rupture of column dominance.

Process Scheme 9 depicts the synthesis of compound (12A) described in Applicants' Example XVIII. are doing.

Summary of the invention Hydrolytically effective oligodeoxynucleotide conjugates of the invention are herein defined as is the desired organic molecule expressed as a ligand, and the metal ion that confers hydrolytic activity. and the desired oligodeoxynucleotides. Effective of the present invention The conjugate has two or more imidazole groups that impart cleavage activity to RNA and one or more imidazole groups. from the desired nucleoside, nucleotide or oligodeoxynucleotide of It has become.

In one embodiment, the present invention provides a metal complex effective for hydrolyzing RNA and a nucleoside. metals covalently bonded to nucleotides and oligodeoxynucleotides. Based on the preparation of complexes. Nucleosides, nucleotides and oligodeoxy By means of a metal complex covalently bonded to a nucleotide, the present invention can be applied to the above C, A, 5t The reasoning of Ein et al. and the teachings of P. G. Schultz et al.

5tein et al. proposed that nucleosides and oligodeoxynucleotides Certain compounds containing imidazole attached to It was found that it is not effective in hydrolyzing RNA. These compounds and RN The lack of A hydrolytic activity was demonstrated in compounds (24), (25) and (26) as It is shown in Table I.

Agents as used herein include metals and ligands or oligodeoxynucleotides. leotide and the conjugates and oligodeoxy conjugates as defined herein covalently bonded to the conjugates and oligodeoxy conjugates. refers to a hydrolyzed compound of the synthetic RNA of the present invention consisting of a metal complex as defined above. O Rigodeoxynucleotides can be used to modify the sequence of an RNA target under physiologically favorable conditions. therefore, it provides a sense of control. The drug of the present invention contains natural glue bonuclease and It is an effective artificial enzyme similar to lipozyme. These drugs are sequence-dominated R compared to ribonuclease and lipozyme in applications where NA hydrolysis is desired. It has several advantages. These advantages include (1) over ribonucleases; High specificity, (2) increased chemical stability over lipozyme, (3) standard chemistry (4) ease of production and isolation by conventional techniques; (4) ease of production and isolation for any targeted RNA strand; (5) low molecular weight, (6) drug transport, and (7) It has the ability to control the hydrolysis activity by polarizing the properties of metal complexes. Ru. These are important aspects of the invention and include sequence-directed RNA hydrolysis and cleavage. can be actually applied. These aspects of the invention incorporate the teachings of the prior art, examples For example, the above-mentioned universal patents, incorporated, PCT special Comparison with the patent application and the documents of P, C, 5chultz et al. and C, B, Chen et al. offers considerable novelty and advantages compared to

5Chult Z et al.'s nucleic acid hydrolysis compound differs from the present invention in several important respects. ing. The nucleic acid cleavage behavior provided by enzymes taught by 5chuitz et al. However, the synthetic low-molecular hydrolyzing agent (artificial enzyme) used by the applicants Therefore, it will not be provided. These enzymes remove proteolytic components from other enzymes. easy to understand. The activity of staphylococcal nuclease is determined by the amount of added calci. It changes depending on the um. Ribonuclease S is S- derived from ribonuclease 8. It is a non-symbiotic complex consisting of a protein and an S-peptide. This complex is easily dissociated cleavage efficiency and specificity. Oligodeoxynucleotide stuffy Rokotsu nuclease conjugates have been shown to cleave DNA as well as RNA. This lacks the specificity of our drug for RNA hydrolysis and cleavage only. Ru. This high activity allows 5chultz The specificity of the enzyme-based system developed by et al. these enzymes The specificity of systems based on , 0°C).

From some practical points of view, the brittle polymer described by 5chultz et al. The molecular weight enzyme-oligodeoxynucleotide conjugates of the present invention are low molecular weight, stable This is a significant disadvantage compared to synthetic ribonuclease analogs.

Some practical aspects of the invention include the ability to prepare more than trace amounts of drug: The ability to prepare pure substances free of active or unknown inactive impurities Ability to remain viable under in vivo conditions and evade immunological responses and DNA and RN It has the ability to avoid nonspecific hydrolysis of both A and A.

Use of hydrolysis according to the invention as a chemical reaction to cleave RNA and more than one By using the imidazole group of RNA and D It offers several advantages over prior art techniques such as non-selective oxidative cleavage of NA. It will be done. The hydrolyzing agent of the present invention is active at pH 7, which is consistent with conditions inside living cells. Ru. DNA is chemically hydrolyzed at a much slower rate than RNA, so this Sequence-directed hydrolysis of RNA using oligodeoxynucleotide conjugates of the invention At appreciable rates their own oligodeoxy-nucleotide components are cleaved. Not done. See Example II of the invention below.

As used herein, the term oligodeoxynucleotide includes oligodeoxynucleotides. xynucleotides, and e.g. Watson-Crick or Zubustin salts Oligodeoxynucleotide analogs useful for group-based molecular recognition are included.

Examples of such oligodeoxynucleotide analogs include alkylphosphotriates. Non-ionic compounds such as esters, alkyl phosphonates and alkyl phosphoramidates Those with onionic internucleotide bonds (P, S, Miller, IDIO) Oligodeoxynucleotide antisense inhibitors of type expression (Oligonu cleotides antisense [nhibitors of Gen e Expression), J, S.

Supervised by Cohen, CRC Press, Poka Sea Tong, FL, 1989, Chapter 4 and references therein), and phosphorothi Contains sulfur-containing internucleotide bonds such as oleate and phosphorodithioate Compounds (C, A, described in 5tein et al., ibid., Chapter 5 and the literature therein), and Alpha oligodeoxynucleotide (B, Rayner et al., ibid., Chapter 6) and its literature). Other suitable oligodeoxynucleotide analogs are carbonates, acetates, carbamates, dialkyl and diaryl silicones. Some have internucleotide bonds such as lyle groups.

Metal compounds applicable to the present invention are water-soluble at neutral pH and physiologically suitable. Metal complex conjugates and olefins that are functionally effective for hydrolytic cleavage of RNA under conditions It is a oligodeoxynucleotide conjugate. In its active form, it hydrolyzes RNA. The metal complexes may contain hydroxyl or aqualigands or both.

These active forms can be prepared using standard methods such as chloride, bromide, iodide, and perchlorate. salts, nitrates, sulfates, phosphines, phosphites and other standard mono- and bi- Complexes containing auxiliary ligands such as bidentate ligands can be induced from The metal of the metal complex can be any metal that is effective in hydrolyzing RNA. Can be metal. Typical metals include copper, zinc, cobalt, nickel, Palladium, lead, iridium, magnesium, iron, molybdenum, vanadium, These include thenium, bismuth, magnesium, rhodium and lanthanide metals.

Measuring agents that can be evaluated in the art, such as the metal complexes and conjugates of the present invention. In order to provide a method for We developed a method to observe RNA hydrolysis at 37°C.

In another aspect, the invention provides: (1) two or more imidazole molecules for RNA clefts. (2) nucleosides, nucleotides and oligodeoxy It is based on the preparation of conjugates containing imidazole groups co-linked to nucleotides. . Proposed by 5tein et al., nucleotide and oligodeoxynucleoly Certain compounds consisting of a single imidazole attached to a tide have been prepared and used in accordance with the present invention. found that it is not effective against RNA under these conditions.

Assay Method of the Invention for Hydrolysis of RNA Adenylic Acid Oligomers of Length 12 to I A mixture of 8 nucleotides [poly(A) +2-+*] as a substrate for analysis use Individual cleavage products from substrate fragments using ion-exchange HPLC divide things. As shown in Figure 1, a sample performed under the same conditions in the absence of a cleavage agent. The compound hydrolytically degrades the substrate to a greater extent than observed in control reactions. When a solution is given, the compound is assumed to be active. The degree of reaction is at time M = 0 It is determined from the ratio of the integrals of the substrate peaks during and time = 18 hours.

HPLC analysis was performed using a Waters 600 multisolvent transport apparatus and A 490 programmable multi-wavelength detector is used. data is water Using Waters Maxima 820 software NECAp c rv Advanced Personal Computer (Advan) ced Personal Computer). using this device Then, it is possible to measure the area under all substrate peaks. meticulous standard Care must be taken to avoid RNase contamination and all buffers must be diluted with prepared with chill-pyrocarbonate treated water (0.1% vol/vol) and the reaction Perform in lopylene tubes. RNA [poly( A) +*-1e] A typical stock solution is 20mM HEP with p)(=7.1 Prepared by dissolving RNA10 units in ES buffer. HPLC analysis is 7 μM Nucleogen DEE AE 60-7 Above, solvent A = 20mM KHz POa, 20% acetonitrile, pH = 5. 5 and solvent B = solvent A + lNKCl, 0-15 min 25% B, 15-4 Perform the elution gradient end from 5 minutes 60% B145 to 60 minutes 100% B.

Combinations of drugs applicable and useful in the present invention are measured by this analytical method. It functionally promotes RNA hydrolysis. The above analysis method is a limitation of the present invention. You shouldn't think so. Other analytical methods can be developed and used to perform the addition of RNA according to the present invention. The effect of water-splitting agents can be measured.

Further consideration of the effectiveness of metal complexes for hydrolyzing RNA is the formation of conjugates. Ru. In forming such conjugates, the selected ligand is first attached to the desired nuclease. Ligands selected metal ions after co-conjugation to osside or nucleotide can be combined with Alternatively, complete selected metal complexes can be converted to nucleosides or or nucleotides. Ligand or complete metal complex can be covalently attached to a nucleoside or nucleotide at any site . Details of the formation of specific conjugates are described in detail in this example.

Similarly, in preparing the oligodeoxynucleotide conjugates of the invention, the selected The selected ligand is first coupled to the desired oligodeoxynucleotide, and then the selected ion can be bound to the ligand. Alternatively, complete selected gold A genus complex can be co-conjugated to an oligodeoxynucleotide. Riggan The compound or metal complex can be co-conjugated to the oligodeoxynucleotide at any position. can be done. Details of the formation of specific oligodeoxynucleotide conjugates The details are described in Example XIV.

Example The examples below demonstrate specific metals, imidazole groups, ligands, and nucleosides. , the present invention relating to nucleotides and oligodeoxynucleotides (their composition, method and utility). The specific contents described in these examples are as follows. Regarding the scope of the invention or applicable agents, elements or features of the invention It should not be considered a limitation.

Example 1 In this example, metal complexes and other compounds were tested for RNA hydrolytic activity. The screening method is shown.

Stock solutions (ImM) of various transition metal complexes (shown in Table 1) were prepared at 2001M Prepared in HEPES buffer pH=7.1. Analytical mixture with final volume of 1.5 ml The compound contained 133 μM metal complex, 63 μM poly(A) 1□-1, and 20 dHEP. Contained ES buffer. At time 20 hours, remove 200 μl of the mixture and It was analyzed by the analytical method of the present invention. The reaction mixture was then incubated at 37°C for 18 hours. After incubation, a second 200 μl was taken and analyzed. RNA hydrolysis Active and inactive transition metal complexes in solution and obtained by measurement using the analytical method of the present invention. The corresponding cleavages are summarized in Table 1.

Active cleavage % Inactive cleavage % Cu(trpy)” 75 Cu(bpy)” 0Cu(bpY)” 43N 1-(CR)” OZn-N-Me-(CR)” 20 [CuCR(CHt) *CuCR]"0Cu-2,2-CR"22Cu(EDTA)0Zn( EDTA) 0 Zn (NTA)' -0 Abbreviation: trpy-2,2': 6', 2-terpyridine; bpy-2,2 -Bipyridine; N-Me-(CR)=7-(N-methyl)-2,12-dimethy Ru-3,7,11,17-tetraazabicyclo[11,3,1]butadeca-1 (17), 2.11.13.15-pentaene, CR=2.12-dimethyl- 3,7,11゜17-tetraazabicyclo[u,3.1]butadeca-1(17 ), 2.11.13.15-pentaene; 2.2-(CR)=2.10-dime Chil-3,6,9,12-tetraazabicyclo[9,3,1]pentadeca-1 ( 15), 2.11.13.15-pentaene, EDTA=ethylenediamine Tetraacetic acid; Nidradotriacetic acid; CRCCH2) sccR>=7.7° (1,3-propylene) lopandiyl)-bis[2,12-dimethyl-3-7.11.17-tetraaza Bicyclo[11,3,I]heptadeca-1 (17), 2. Il, 13°15 -Pentaene.

Table 2 shows that the analytical method of the present invention was not found to be effective in hydrolyzing RNA. Compounds known to the present applicants are summarized.

The identity and structure of the compounds in Table 2 are shown in FIG.

Example II In this example, conditions under which the Cu(bpy) complex substantially hydrolyzes RNA are described. Below, it is shown that DNA is not degraded.

The observed RNA cleavage by various types of Cu(bpy) is hydrolytic. , was not oxidative. This is a combination of Cu(bpy) complex and DNA and RNA. This was shown by comparing the reactivity with

DNA [Poly(dA)I□-1,1 stock solution, 25 units of DNA at 20mM Prepared by dissolving in HEPES buffer, pH=7.11.0+nl . The reaction mixture contained 63 μM DNA, 157 μM bipyridine in a total volume of 1.5 ml. contained 157 μM CuCL and 20 mM HEPES buffer. melt After incubating the solution at 37°C for 48 hours, it was analyzed by ion-exchange HPLC. analyzed. The same conditions were applied to Cu(bpy)” complex and RNA [poly(A)+z- +m].

Figure 6 shows the HPLC fraction of the reaction between the Cu(byp)”+ complex and DNA and RNA. It shows the analysis. After 48 hours, the RNA is extensively hydrolyzed. in contrast, The DNA substrate showed no signs of degradation. RNA and DNA move at the same rate 1, 10-phenanthroline-copper (reported to be oxidatively cleaved by m (C, B, Chen et al., J. Am, Chem, Sac, 1988. 110.6570-6572), therefore, if the oxidative mechanism works, It is expected that a radical cleavage of DNA by the Cu(byp)+ complex will be observed. Ru.

Example III This example shows that the 3' of the 2'-deoxy-thymidine nucleotide shown in step @l The binding of bipyridyl ligand (bpy) to the position shown in FIG.

5'-0-DMT-2'-deoxy-thymidine-3'-0-q-cyanoethyl- N,N--diisopropylphosphoramidite (1) (0.4 g, 0.537 mi Tetrazole (remol) was dissolved in anhydrous CHsCN (5 ml) under N2, 0.112 g (51.61 mmol) was added.

DMT is 4,4'-dimethoxytrityl. The resulting mixture was heated at room temperature for 15 minutes. Stir for a while. 4'-Methyl-4-(hydroxybutyl)-2,2'-bipyridine (2) (0.130 g, 0.540 mmol) in acetonitrile solution (5 ml) was added and after 1 hour the mixture was concentrated in vacuo to give a glassy compound 5'-〇 -DMT-2'-deoxy-thymidine-3'-0-[4-[4'-methyl( 2,2'-bipyridin)-4-yl]butoxy]-q-cyanoethoxyphosph fin (3) was produced. Compound (3) was cooled to 0°C in a water bath in CH2C1 i (3 ml) and add t-BuOH to 2. 2. 4. 4-tetramethyl A solution in pentane (0,643ml, 1.93 mmol) was added. 20 After minutes, the mixture was concentrated in vacuo to form a glassy compound 5'-0-DMT-3'- [4-[4'-ethyl(2,2'-bipyridin)-4-yl]butyl]-q-cy Anoethylphosphate)]-]2'-deoxythymidine 4) was produced. Compound (4) (0,382 g, 0.432 mmol, 80.4%) is 5% M e O Eluted from the alumina column (neutral) using H/CHtCI2.

Compound (4) (0,382 g, 0.432 mmol) was dissolved in aqueous NH3 (10 ml) and stirred at room temperature for 6 hours.

The mixture was concentrated in vacuo with EtOH to remove water. 25% CFi of the residue Treated with C0OH/CHtCL (5 ml) for 15 minutes. volatile components in vacuum After removing the residue, the residue was dissolved in water (10 ml) and the aqueous layer was dissolved in ether (2 x 5 m l) and CH2CL (2g5ml). Concentrate the aqueous layer in vacuo and Desired deprotected nucleoside 3'-[4-[4'-ethyl(2,2'-bipi lysine)-4-ylcobutyl-phosphe-)]-]2'-deoxythymidine Ammonium salt 5) (0,192g.

0.354 mmol, 82%).

Example TV In this example, compound (5) was titrated with 1 g of CuC, and 3'-[4-[4'- Methyl(2,2'-bipyridine)-4-ylcobutyl-phosphate)]-]2' -deoxythymidine=ammonium salt copper II) (Figure 2) are doing.

Compound (5) was dissolved in 20mM HEPES buffer at pH 7.1 at 53.4μM. Place 1 ml of the solution in a quartz cell and add a 1,178 mM stock solution of CuCL dissolved in water. Added a few drops of liquid. Changes in the visible spectrum were observed at wavelengths from 240 to 380 nm. . With the addition of CuCIz, the 276 open band decreases and the 302 and 31 Impurities at 2 nm absorbance increased. This change is due to the iso-isolation at 289 nm. This occurs due to isosbestic pint, and the This is a characteristic of coordination to pyridine.

Similarly titrated with a solution of 3'-thymidine monophosphate without the bipyridine ligand. However, no changes were observed in the visible spectrum over the mentioned region.

In this example, R Figure 3 shows the hydrolysis of NA [poly(A) +2-+-].

For the reaction of metal complex-nucleotide (nucleoside) conjugates, the applicant et al. The HPLC analysis method was modified.

These compounds do not have high reactivity as free metal complexes, so the reaction time is 48 hours. It was extended to

In a total volume of 50 μl, the reaction mixture contained 157 gM of compound (5), 63 μl of RNA M, Cu(SO4) 157μM and 20mM HEPES buffer, pH=7. It contained 1.

Using this mixture, compound (6) was formed under the conditions described in Example IV.

01li? Between 100 μm and 100 μm of the reaction mixture, Applicants' HPLC analysis Immediately analyzed by analytical method. Incubate the reaction mixture at 37°C for 48 hours. After sampling, a second sample was removed and analyzed. The RNA substrate is prepared by compound (6). It was found that it was clearly hydrolyzed (Fig. 3).

In a control reaction performed in the same manner except for the absence of Cu (SO=), 48 No RNA hydrolysis was observed after hours of incubation.

Example VI In this example, the 2'-dehyde shown in Process Diagram 2 for bipyridyl ligand (bpy) is The bond to the 5' position of oxythymidine nucleoside is shown.

5'-[4-[4'-methyl(2,2'-bipyridin)-4-yl]butylco -methylphosphene)-3'-0-acetyl-2'-deoxythymidine (10) Preparation of 4-[4'-methyl(2,2'-bipyridine)-4-ylcobutyl-methyl Chill-N. N--Diisopropylphosphoramidite (8) (0,101g, 0 ．． A mixture of 25 mmol) and tetrazole dissolved in 1 ml of THF was prepared at room temperature for 10 min. Stir for a minute. 3'-0-acetyl-2'-deoxythymidine (7) (0,07 1g, 0.25 mmol) dissolved in CH, C1t (1 ml) was added to the reaction mixture. and the solution was stirred for 60 minutes. The mixture was then filtered to remove the precipitated material. Torazol was removed. The solid was purified with acetonitrile (5 mD and CH2C1x (5 ml) and concentrated to give a glassy 5'-0-[[4-[4'-methylene] (2,2'-bipyridin)-4-yl]butoxy]methoxy]-3'-0-a Cetyl-phosphine-2'-deoxy-thymidine (9) was obtained. of mixture (9) The glassy material was dissolved in MeOH (tml), cooled to 0°C, and then dissolved in t-butylhydroxide. Droperoxide 2.2.4,4. -tetramethylpentane (0.3 ml, 0 ．． 9 mmol) was added to the stirred reaction mixture. 15 minutes later , the water bath was removed and the mixture was stirred at room temperature for 20 minutes.

The mixture was concentrated to a glassy material and flashed onto an alumina column (TLC grade). Shuchromatography was performed.

Desired compound (10) (0,071 g, 0.118 mmol, 47%) as eluent As a group from CHt CI t to 10%Me OH/CH* Cl t The column was eluted using Radiend.

5'-[4-[4'-methyl(2,2'-bipyridine)-4-ylcobutylpho Sphateco-2'-deoxythymidine-triethylammonium salt (11) gII compound (10) (0,071 g, 0.118 mmol) at CHZC 25 in which NaOMe was dissolved in M e OH was dissolved in L (3n+1). % solution (0.05ml 10.12 mmol) was added. Stir the mixture at room temperature for 15 minutes. Stirred.

After 15 minutes, the reaction mixture was quenched with glacial acetic acid (0.06 g, 0.12 mmol).

Dichloromethane (50ml) was added and the organic layer was dissolved in saturated NaHCO3 (2X20ml). ) and water (10 ml). The organic layer was intercalated over anhydrous Na2SO4 and Concentrate in vacuo to give a glassy substance (0°064 g, 0.114 mmol, 97%). Ta. This glassy material is manufactured by Sigma Chemicals. ), a commercially available deprotection reagent, thiophenol:dioxane:trinitylamide. (1:2:2) and stirred for 90 minutes. Concentrate the mixture in vacuo The residue was dissolved in water (1'0 ml) as a glassy substance. The aqueous layer is replaced by petroleum Traces of thiophenol were removed by washing with ether (2xlOml). Final Purification was performed using a RP C-18 Sep-Pak column. , desired compound (11) (0,069 g, 0,106 mmol, 90%) at Hz0 :CH3CN (4:1).

Example VII In this example, compound (11) prepared in Example Vl is titrated with CuC1x. 5'-[4-[4'-methyl(2,2'-bipyridine)-4- yl]-butylphosphate)]-]2'-deoxythymidine triethylane Formation of monium salt #(IrXI2) is shown.

The procedure described in Example II was followed. Characteristic of the coordination of copper (I1) to bipyridine Changes in the visible spectrum similar to those shown in FIG. 3 were observed. Thymidine-5 For the titration of '-monophosphate, the visible spectrum over the range 240-380 nm is No change in torque was observed.

Example VIII In this example, a nucleotide covalently bonded to a transition metal complex via the 5' position RNA [Poly (A) +! -1m].

As described in Example Iv, except that compound (12) is a hydrolyzing agent (Figure 4). The same procedure was used.

Example Ix In this example, the uracil 5 in the uridine nucleoside shown in Process Diagram 3 is Binding of bipyridyl ligand (bpy) to the - position is shown.

5'-0-DMT-5-[3-[[4-[4'-methyl[2,2'-bipyri gin]-4-yl]-1-oxbutylcoamino]ethyl]amino]-3-oxy Preparation of Sobrovir]-2'-deoxy-uridine (15)+5-[3-[(2- aminoethyl) aminoco-3-oxophenyl'J-5'-0-DMT-de Oxy-uridine (13) (0,322 g, 0.5 mmol) was dissolved in CH3CN (5 ml) and Et3N (0,’2 ml) in a water bath at 0°C. 4-[3-carpo-3-(p-nitrophenoxy)propyl]-4' -Methyl-2,2'-bipyridine (14) (0,566 g, 1.5 mmol) Added to stirred reaction mixture. After 15 minutes, remove the water bath and leave the mixture at room temperature for 24 hours. Stir for a while. The reaction mixture was mixed with CHz C1x (20 ml) and water (10 ml). diluted with

The aqueous layer was washed with CH2Cl□ (2X20ml). dried (MgSO4) Concentrate the organic layer in vacuo and flash chromatograph on an alumina column (neutral) Gradier from CH2C1f to 6%EtOH/CH2C] p-nitro-phenol was removed. The desired product was dissolved in EtOH was used to elute from the column with c-3bpy acid. Compound (15) (0,2 11 g, 0.24 mmol, 45%) by RP HPLC at a flow rate of 6 ml/ Purification was performed using a linear ternary gradient of minutes. Solvent A (0,1M) [Et 3NH] MeCN and 820 were varied while OAc was held constant.

5-[3-[[2-[[4-[4'-methyl[2゜2'-bipyridingo4- yl]-1-oxobutyl]amino]ethyl]amino]-3-oxopropylgo Preparation of -2'-deoxy-uridine (I6): Nucleoside (15) (0,1 CF *Treatment was carried out for 15 minutes with a 10% solution (5 ml) of C0OH dissolved in CH, C1t. . The mixture was concentrated to a glass and dissolved in water (10ml). aqueous layer Washed with CH, C1* (2X fail) and obtained compound (16) (0,093 g.

0.16 mmol, 94%) at a linear ternary gradient with a flow rate of 6 ml/min. Purified by RPHPLC using Solvent A (0,1M) [Et! N H] Hold OAc constant, M e CN and H! O was changed.

Example X In this example, 5-[3- [[2-[[4-[4'-methyl(2,2'-bipyridin)-4'-yl]-- 1-oxobutyl]amino]ethylcoamino]-3-oxopropylgo2'- Figure 2 shows the formation of deoxy-uridine steel (II) (17).

The procedure described in Example III was followed. Copper (m bits) similar to that shown in Figure 3. A change in the visible spectrum characteristic of coordination to pyridine was observed. drops of uridine Under normal conditions, no change was observed in the visible spectrum over the 240-380 nm range. Ta.

Example XI In this example, co-linkage is performed via the 5-position of uracil in the uridine nucleoside. Figure 2 shows the hydrolysis of RNA [poly(A) +2-+s] by gold aS.

Same as described in Example IV except that compound (17) is a hydrolyzing agent. procedures were used. Hydrolysis of RNA is evident as shown in FIG.

Example XII In this example, the nucleotides described in Examples IV and V can be attached. , each previously shown to be an active RNA hydrolysis catalyst in Example I. Figure 4 shows the preparation of seed terpyridine (tpy) derivatives (Scheme 4).

4'-(3-formylpropyl)-2,2': 6'+2#-terpyridine ( 19) Preparation: rubber stopper, Teflon-coated stirring rod and gas intake adapter A 100 ml three-necked RB flask equipped with a adapter was purged with N. described in the literature Methods (K, T, Potts et al., J, Am, Chem, Soc, 19 87, 109.3961-3967)). Chill 2.2': 6'+ 2#-Terpyridine (18) (0,494g, 2ml mol) was dissolved in dry THF (10 ml) and poured into a flask. reaction mixture Cool to -78°C and add LDA (1.6 mm, 2.4 mmol) to the syringe. Added via. The resulting dark brown mixture was stirred at -78°C for 2 hours. 2-(2 -bromoethyl)-1,,3-dioxolane (0,434 mL 2.4 mmol ) into the reaction mixture, stirred for 1 hour at -78°C, and incubated for -night. It was then brought back to room temperature. The mixture was poured into 10 ml of brine, and the aqueous layer was dissolved in CH, CI, Extracted with. The extract was dried over MgS Oa and evaporated to dryness to yield the crude acetate. Got tar. This acetal was mixed with IMMCl (10+ol) at 50-60% Hydrolysis was carried out by heating at °C for 2 hours. The solution was then diluted with aqueous N a H Neutralized with COs and extracted with CHiC1*. Dry the extract over MgS O4 and evaporated to dryness to obtain the crude aldehyde. Flange chromatography (neutral Alumina, CH, CI! The pure aldehyde (19 ) (0,339 g, 1.12 mmol, 56%).

4'-(4-hydroxybutyl)-2,2':6'.

Preparation of 2'-terpyridine (20): Aldehyde (19) (0,303 g, 1 ml Dissolve sodium borohydride (0, 05 g, 1 mmol) was added at room temperature. After stirring for 30 minutes, the mixture was of brine and the aqueous layer was extracted with CHzc1□ (3×10 ml). extract liquid was dried over MgS OJ and evaporated to dryness, resulting in the desired alcohol (2 0) (0,264 g, 0.86 mmol, 86%) was obtained.

Compound (20) is similar to compound (2) in Process Schematic 1 and is described in Example II. It can be attached to the 3' position of thymidine in the same manner as described above.

4'-(4-bromobutyl)-2,2': 6',2''-terpyridine (21 ) Preparation of alcohol (20) (0,200 g, 0.65 mmol) in HBr ( 45%) and refluxed for 6 hours. After cooling to room temperature, scrape the mixture. Pour into 20 g of ice, make basic with a saturated solution of NaC0□, and extract with CHICl. Ta. When the extract was dried on MgS OJ and evaporated to dryness, the bromo derivative (21) (0.186 g, 0.51 mmol, 78%) was obtained.

4'-(4-phthalimidobutyl)-2,2': 6'+2#-terpyridine Preparation of (22): Bromo compound (21) (0,186 g, 0.51 mmol) Potassium phthalimide (0,095 g, 0 ．． 51 mmol) was added to a suspension in DMF (1 mmol). Mixture 50-6 Stirred at 0°C for 2 hours. After cooling, the reaction mixture was poured into water (10ml) to The mixture was thoroughly extracted with CHC1s (3 x 25ml). Combine the organic layers , 0.2M NaOH (201+11), washed with water (20ml), Na25OJ Dry on top. When the solvent was distilled off under reduced pressure, a viscous oily product was obtained.

When recrystallized from ethanol, compound (22) (0, 208 g, 0.48 mmol, 95%), melting point 128°C.

4'-(4-aminobutyl)-2,2':6',2''-terpyridine (23) Preparation of phthalimide derivative (22) (0,208 g, 0.48 mmol) in E Suspended in OH (7 ml), added hydrazine hydrate (88 mg, 0.48 mmol) Processed with The mixture was refluxed for 6 hours, cooled to room temperature and diluted with brine (IO ml). and basified to pH 12 with 50% (wt/wt) NaOH. mixture The material was thoroughly extracted with CHIC It (3×10 ffll). Nail the organic layer Dry over SO4. When the solvent was distilled off under reduced pressure, the desired product (23) ( 0.130 g, 0.43 mmol, 89%).

Compound (23) was prepared at the 5'-position of 2'-deoxy-thymidine by procedures described in the literature. (B, C.

F., Chu et al., DNA, 1985.4.327-331).

Example XIII In this example, oligodeoxynucleotides according to the present invention shown in Process Diagram 6 were prepared. Sequence-directed cleavage of tRNA'' by di-bipyridine steel (II) (32) complex show.

101 μM stock solution of oligodeoxynucleotide-bipyridine conjugate (31) is compound (31)6. 1 unit of 20mM HEPES buffer pH 7.1 50 It was prepared by dissolving at 0 μm. 25.9 μM stock solution of tRNA”’ substrate The solution is 10 units of tRNA in 20mMHE PES buffer at pH 7.1. Prepared by dissolving in 500 μl. The cleavage reaction occurs when the total amount is 600 μm. , 1.29gM tRNA "γ', l 2.9μMCu (trpy)", 2 27gMCu (SO4), 6.4ttM compound (31), 50mM NaC! , and 50 mM HEPES buffer at pH 7,8. First, tRNA ”’, Compound (31), NaCl and buffer were combined and heated to 65°C in a water bath for 4 hours. Heated for minutes. The reaction was removed and immediately placed on dry ice. blend After melting at 0 °C, Cu (So, ) and Cu (trp’l)” complexes were added. Eta.

Applicants demonstrate in Example III that copper ([I[) is arranged on a bipyridine ligand. In this case, the oligodeoxynucleotide-gold lI# conjugate (32) It was shown that it is formed exclusively. Heat the reaction mixture at 37°C and aliquot 100 μl. were removed after time = 0.17 and 28 hours.

When the collected amount was analyzed by polyacrylamide gel electrophoresis, it was found that the 6th Three distinct cleavage sites adjacent to the targeted sequence are revealed as shown in the figure. became.

These bands appear in a time-dependent manner, and the control reaction Regions of 0 or 1 lack cleavage or showed significantly reduced cleavage.

RN based on hybridization of compound (32) to tRNA'' The predicted position of A cleavage resulted in a fragment 51 nucleotides in length (Process diagram) 5). This fragment was observed. Two other fragments also result from the reaction. Ta. These reactions occur in compound (32) due to the three-dimensional folding of the tRNA'' molecule. This is due to cleavage at a site close to the group. Therefore, tRNA” ’ sequence-directed cleavage was demonstrated.

Example XVI In this example, as shown in process diagram 7, the nucleoside (5A) is 5-[3-[[2-[[2-[[2-amino]-1-xo-3-[IH-i midazol-4-yl]propyl]amino]-1-oxo-3-[IH-imida sol-4-yl]propyl]amino]ethylcoamino]-3-oxopropyl ] -2'-Deoxy-uridine-containing di-imidazole synthesis.

The synthesis of compound (IA) has been previously described (Dervan et al., Proc. Natnl, Acad, Sci, USA, 1985.82.968) .

Nucleoside 5'-0-DMT-5~[3-[(2-aminoethyl)-aminoco -3-oxopropyl]-2'-deoxy-uridine (IA) (1,288 g, 2.0 mmol) was dissolved in dry dichloromethane (IO ml) and heated to 0°C in a water bath. Cooled. Fmoc-L-Hi　(Tr)-o-pfp　(3,16g, 3 ．． 0 mmol) was added to the stirred reaction mixture. Triethylamine (0.28m 1°2.0 mol) was added to the solution and the mixture was stirred at room temperature for 8 hours. reaction mixture Concentrate to 100% C by flash chromatography on a silica gel column. H! C1t to 12% Et OH/CHt Cxz (7) Gradient It was purified by elution with Nucleoside 5'-〇-[bis[4-methoxy Cyphenyl)phenylmethyl]-5-[3-[[2-[[2-[[(9H-fluor oren-9-yl-methoxy)carbonylcoamino]-3-[1-(triphenylated) nylmethyl)-1H-imidazol-4-ylcobropyl]amino]ethyl]a [mino]-3-oxobrovir]-2'-deoxyuridine (2A) (1,94 g , 1.56 mmol, 78%) from the column with 10% EtOH/CH,CL. It was eluted using

Nucleoside (2A) (1.94 g, 1 ．． 56 mmol) was treated with Et2NH (IO ml) at room temperature and the mixture was concentrated. It was shrunk to form a glassy substance. Chromatograph the residue on a silica gel column. It was. The deprotected impurities are eluted from the column using CH,C1, and the nucleotides are Sid 5'-0-[bis(4-methoxyphenyl)phenylmethyl]-5-[ 3-[[2-[amino]-3-[1-()liphenyl)-IH-imidazole -4-yl]propyl]amino]ethyl]amino]-3-oxopropyl]-2 '-Deoxy-uridine (3A) (1,43 g 51.4 mmol, 90%) Elution was performed using 80% EtOH/CHI CI. (3A) (1g, 2. 0 mmol) dissolved in dry dichloromethane (IOml) in a water bath. Cool to 0°C in a medium, and add Fmoc-L-His (Tr)-0pfp (3,1 6 g, 3.0 mmol) was added to the stirred reaction mixture.

Triethylamine (0.28ml 1.2.0mol) was added to the solution and the mixture was heated at room temperature. Stirred for 8 hours. Concentrate the reaction mixture and flash chroma it on a silica gel column. 100% CH2Cl to 15% EtOH/CH*C1z It eluted at the gradient end. Nucleoside 5'-0-[bis(4-methoxy) Cyphenyl) phenylmethyl] -5-[3-[[2-[[2-[[2-E] (9H-fluoren-9-yl-methoxy)carbonylcoamino]-1-oxo -3-[1-)liphenylmethyl)-1H-imidazol-4-yl]propyl ]Amino]-1-xo-3-[1-()liphenylmethyl)-1H-imida zol-4-yl]propyl]aminoconityl]amino]-3-oxopropyl ]-2'-Deoxyuridine (4A) (1.94g51.56mmol, 7 2%) was eluted with ■2% EtOH/CH, C1t.

Melting point 156-8°C.

Nucleoside (4A) (0.4g 10.246 mmol) in CH, CI□ (5 ml), treated with diethylamino (5 ml) and stirred for 9 hours. blend was concentrated, and the residue was subjected to flash chromatography on a silica gel column.

Compounds eluted from the column using 10% NHZ/EtOH were treated with ■5% CF, C 0OH to CH, CI.

(5 ml) and stirred. After 30 minutes, the mixture was concentrated. residue It was suspended in CHtC1, (25 ml) and water (IO ml). aqueous layer to Hz Washed with CIx (2 x 5 ml) and ether (2 x 5 ml) and concentrated. However, diimidazole nucleoside conjugate 5-[3-[[2-[[2-[[ 2-aminoco-1-xo-3-[IH-imidazol-4-yl]propyl] amino]=1-oxo-3-[IH-imidazol-4-yl]propyl]amino [ethyl]amino]-3-ogisobropyl]-2'-deoxyuridine (5) (o, 120 g, 0.194 mmol, 79%).

Example XVZ In this example, cleavage of a 172-mer RNA fragment by compound (5A) and mono-imidazole compounds, 5-[3-[[2-[[2-amino-3- (LH-imidazol-4-yl)-1-oxopropyl]amino]ethyl]a Mino-3-oxoprobyl-no-2'-deoxy-uridine (4A) (Bash Kin, Gard and Modak, J.

Org, Chem, 1990.55.5125) under the same reaction conditions This shows that NA fragments are not hydrolyzed (Figure 9).

Great care was taken to avoid contamination of RNase with the hydrolysis reaction. All buffers are , distilled deionized treated with diethylpyrocarbonate (0.1% vol/vol) The hydrolysis reaction was carried out in a sterile polypropylene tube. Cleavage anti Analysis of reactions was performed using denaturing polyacrylamide gel electrophoresis using standard techniques. (D'A1essio, J.M., Practical Guide to Nucleic Acid Gel Electrophoresis ゛Method (In Gel Electrophoresis of Nuclei) c Ac1ds APpractical Approach), D, Rick supervised by Wood and B, D, Hanes, IREL Press Limited ((RI PressLimjted), London, 1982, 173-1 96 pages). Uniformly radiolabeled RNA substrates with 0P can be isolated using standard techniques. Theriophage SP 6 Run-off transfer by DNA-dependent RNA polymerase Mani generated by runoff transcription. atis, T,, Frltsch, E,.

Sambrook, J., Molecular Cloning: Laboratory Handbook (Molecu-1 ar Cloning: A Laboratory Manual), :l -Rudo Spring 11 Harbor Laboratory- (Cold Spring H arbor Laboratory), York, 1982). sigma ・Chemical Company (Sigma Chemical Go,) (St. HEPES Co., Ltd. (Lewis) was used without further purification.

All electrophoresis reagents were RNase-free.

s! Approximately 4011g of P-labeled RNA (7) storage solution was added to 20mM HEP. ES buffer, I) H=7. Prepared by dissolving 1.90 μm. Protection existing solution, (5A)4. 9mM and (6A) 3, 1mM are 20mM Prepared in HEPES buffer. Control reactions (#l) had a total volume of 25 μl. During the loading, stp-labeled RNA diluted in HEPES buffer 4. It contained 5μg. The reaction with drug (5A) was performed in a total volume of 90 μm. It contained approximately 23 μg of recognized RNA and IIIM compound (5A). Drugs ( 6A), approximately 9 μg of IP-labeled RNA and 2 μg of IP-labeled RNA in a total volume of 50 μl. Contained mM compound (6A). All reactions were carried out at 50″C for a total of 2 hours. After that, a 20 μm slice was taken out and immediately frozen on dry ice. Reaction #1 In some cases, a single point was obtained in 2 hours. For reaction #2, aliquot 20μl were taken orally at times = 0, 0.5, 111.5 and 2 hours. For reaction #3 A 20 ml aliquot was taken by mouth for 1=0 and 2 hours.

The specimens were loaded onto a PEGE gel (15%, 7M urea) and run at 900v for 4 hours.

Gels were developed using standard autoradiography methods (Maniatis , T.

Frltsch, E., Sambrook, J., Molecular Cloning: Real Laboratory Handbook (Molecular Cloning: A Laboratory Manual), Cold Spring Harbor Laboratory (Cold Spring Harbor Laboratory), New York, 198 2 years).

The results shown in FIG. Although reaction #1 (lane #1) and #3 (lane #7 and and 8) clearly demonstrate the lack of RNA cleavage. Reaction #3 is 2mM total The fact that it shows no cleavage even at imidazole concentrations indicates that similar compounds like compound (5A) Incorporation of two imidazoles into one molecule prepares for efficient cleavage of RNA It is shown that.

Example XVrlI In this example, imidazole-nucleoside conjugates G, tri and ori Incorporation into godeoxynucleotides is shown. These techniques are suitable for It can be applied to the incorporation of conjugates into oligodeoxynucleotides.

Two complementary methods were investigated for the synthesis of imidazole-DNA conjugates. : Solution phase phosphotriester chemistry and solid loss phosphoramidite chemistry. stomach The method of deviation was previously reported (Baskin, J. Org.

Chet, 1990, 55.5125) Compound (7A), which is compound (6 Use the protected form of A). Therefore, process diagram 8 shows 0-cloning Phenyl phosphate ester method (Reese, C, B, Tetrahe dinucleotide Xpc (compound thing! The use of (7A) in the preparation of OA) is shown. Intermediate phosphodies Tel (8A) was prepared in 82% yield and characterized by FABMS. complete The fully protected phosphotriester (9A) was prepared in 49% isolated yield and F ABMS is M+3L　i, M+2L　i, M+L　i and M+3Li-Boc The corresponding ion is shown. Deprotection of (9A) and reverse phase HPLC of the product After purification, the desired dinucleotide (IOA) was obtained.

Process diagram 9 shows the preparation of phosphoramidite (IIA) and dinucleotide Xpt , showing its use in solid phase synthesis of compound (12A). phosphoramida Ito (IIA) is 149.4 ppm identifiable in its two diastereomers. It also has a characteristic 3IP resonance in FABMS and high-resolution mass spectroscopy. It was characterized by spectra. The identification of the HNMR peak in (15A) is actually The results are shown in the experimental section. - In order to test its general applicability, phosphoramidites 11 is then (13A) an 11-mer oligode which grows a modified nucleoside at an internal position. It was used in the synthesis of oxynuclide. The prepared sequence is 5'-TAT CTTCTXAC-3' (However, X represents an imidazole-containing nucleoside analog. ).

Melting points were determined using a Melt-Temp melting point apparatus with a calibrated thermometer. Measurements above were made with a Kimax soft glass capillary. All nuclear magnetic resonance Spectra were recorded on a Varian spectrometer at 25°C. Ta.

1) (and 13C spectrum was measured on VXR-400, !Ip spectrum was obtained on an XLA-200. Protons obtained by Fourier transform of integral scan The spectrum was 30016 with a spectral width of 8 KHz and an acquisition time of 1.876 seconds. It consisted of data points. The data is a 35° pulse (10ms), 3. Strong H,O resonance if necessary. Presaturated with O8. free induction slow The extension is 32 and 0.5 Hz line applied to the data before Fourier transform. Zero filling in the broadning. Significant chemical shifts p towards below TMS Recorded in pm (in d) and JPC values were obtained in Hz. All compounds have 12C and Purity was >98% according to HNMR spectroscopy. replaceable Protons were labeled (ex).

High-resolution mass spectra (HRMS) were obtained by Finnegan/ Recorded with MAT 90 spectrum analyzer, FAB ten low resolution spectrum It was measured with a vG40-250T spectrum analyzer. The FAB matrix is 3- Particularly useful are saturated solutions of Lil in nitrobenzyl alcohol. It is a protected nucleoside that is acid labile. Baker thin layer chromatography - Performed on Flex Silica Gel IB2-F plate, UV (254 nm) The spots were visualized by irradiation.

Preparative TLC uses a silica gel plate (Analtech). Chromatotron (Harrison Research) The analysis was carried out by centrifugal TLC on RISON Research). column Chromatography on silica gel (Merck 5G-60, 230-240 mesh) I went above. RPHPLC is an automatic method for di- and tri-nucleotides. AlltechEconosil C18 Preparation Column ( 10 m, 250 x 22.5 mm) and a linear ternary gladiator with a flow rate of 6 ml/min. Solvent A (o, 1M [Et, NH]0Ac) at 25% B (MeCN) and C (HaO) were kept constant and varied as follows. Time The interval is in minutes, (hour, %B1%C) (0,5,70)(33,35,40)( 45, 70, 5). Longer oligodeoxynucleotides are nucleogens. - Linear binary gradient with flow rate of 1.5 tnl 1 min on DEAE60-7 preparative column Solvent A (80% 0.1M Na0Ac, 20% Na0 Ac) and solvent B (20% MeCN, 80% IMLiCl and 0.02MN a0Ac) was changed as follows. The time is in minutes, (hours,%A1%B)( 0,100,0)(33,0,100)(43,100,0). HPLC was observed simultaneously at 260 and 400 nm.

p-nitrophenol, L-histidine (Sigma Chemicals), Fmoc- L-Lys (Boc)-Opfp (Pharmacia) , dicyclohexylcarbodiimide, diethylamine, diisopropylethyl Amine (Aldrich), chloro-N.

Like N-diisopropylamino-q-cyanoethoxy-phosphine (ABN) The compound was used without further purification.

5'-0-[bis(4-methoxyphenyl)-phenylmethyl]-5-[3 -[2-[[2-[[(9H-fluoren-9-yl-methoxy)carbonyl] Aminoco-3-[[1-(2,2-dimethylethoxy)carbonylco-IH-I midazol-4-yl]-1-oxopropyl]amino]ethyl]amino-3- oxopropyl]-3'-[o-chlorophenylphosphodiester]-2'- Preparation of deoxy-uridine (8A) 0-chlorophenylphosphodichlororesort (0,897 g, 3.66 mmol) were weighed into two Ronas-type flasks and dissolved in acetonitrile (10 ml).

1.2.4-) Riazole (0,556 g, 8.052 mmol) and Torie Thylamine (1,02 mL 7.32 mmol) was added to the reaction vessel and the mixture was brought to room temperature. Stir at room temperature for 20 minutes. Nucleoside (7A) to acetonitrile (10ml) Dissolve and stir 1-Me-imidazole (0.1 ml, 4.88 mmol) into the solution. added to. Add this reaction mixture to the phosphorylation mixture in the eggplant flask and Stir at room temperature for 20 minutes. The reaction was observed by TLC and after all the starting material was consumed, The mixture was mixed with triethylamine (3.06 mmol, 21.96 mmol) and water (1 0 mm) to make a homogeneous solution. After stirring the solution for 10 minutes, Concentrated. The residue was dissolved in dichloromethane (25 ml) and saturated NaHCO* (25ml).

The aqueous layer was washed with dichloromethane (2 x 20 ml) and the combined organic extracts were washed with Mg Dry over S OJ and concentrate to a glassy material. dichloro glassy substance Dissolved in methane (10ml) and precipitated from petroleum ether (500ml). solid Phosphodiester (8A) (1.16 g, 0.91 mmol, 82%) was centrifuged. It was collected by separation and dried in a vacuum desiccator. FABMSSm/z 117 1, (MH); 1071. (M-H-Boc).

Preparation of dimer X' I) C' (9A) Phosphodiester (0,427 g, 3.36 mmol) was dried and dissolved in lysine (5all), 4-N-3'-0 -Diacetyl-2'-deoxycytidine (0,095g).

3.05 mmol) was added thereto and the pyridine was removed under reduced pressure. of pyridine Two addition and removal steps were performed to remove traces of water. Next, ■−(2− Mesitylenesulfonyl)-3-nitro1.2.4-)lyazole (0,361 g, 12.2 mmol) to a solution of the two nucleosides and at room temperature Stir for a minute.

The mixture was then quenched with 1 ml of N a HCO* saturated solution. 5 minutes Later, dichloro-methane (100 ml) was added to the reaction mixture and the organic phase was diluted with water (50 ml). washed with millimeter).

The organic extract was dried over MgS OJ and concentrated to produce a glass.

This glassy substance was chromatographed on silica gel to produce the product (9A) ( 0,220 g, 1°5 mmol, 49%) in CHz Cit: E tOH= It eluted at a ratio of 90:10. FABMS, m/z 1484, (M+3Li); 1 478. (M+2Li);1472. (M+Li); 1384. (M+3Li -Boc).

Preparation of XpC(IOA) The fully protected dimer (9A) (0.220 g, 0.15 mmol) was added to Nl , Nl, N2, Nl-tetramethylguanidine (0,33M) and 0 - Nitrobenza Ludoxime dissolved in dry CH3CN (1.5 ml) It was treated with a solution prepared in After 3 hours at room temperature, the mixture was concentrated and the residue was purified with ether. Washed with water. The solid was dissolved in aqueous NH, and stirred at room temperature for 24 hours. solution After concentration, the resulting solid was mixed with 50% CF*C0OH in dichloromethane (10% The cells were treated for 30 minutes with a solution dissolved in Completely deprotected nucleosides are diluted with water. (10 ml) and the aqueous layer was washed with diethyl ether (2x 5 ml). Ta. The aqueous layer was evaporated and the deprotected nucleoside (IOA) (0,110 g, 0 ．． 143 mmol, 95%) was obtained.

This sample was prepared using Alltech Econosil C1. Purified on a 8 preparative RPHPLC column. (IOA) (2500D units) Retention times were C18 using the same linear ternary gradient end with a flow rate of 1.5 ml/min. The time for the analytical column was 14.6 minutes. "PNMR (D, O') ppmO, 19s ;' H(Dt 0) d6.25(t, IH, l'X); 6.3(t, I H, I'C): 2.45 (m, 2H, 2'X): 2.4 (m, 2H , 2°C); 4.8 (m, IH, 3’X); 4.6 (m, IH, 3” C); 4.1 (m, IH, 4'X); 4.0 (m, IH, 4°C); 3.8 (m, 2H, 5'X); 4.2 (m, 2H.

5'C): 6.1 (d, 18.Hl9); 7.9 (d, 18.8 18): 7.7 (s.

IH, H6): 2.4 (2t’s, 4H, 87 &H8); 3.2 ( 2t’s, 48°H9 &HIO); 4.25 (m, IH, Hll); 3.2 (m, IH, Hl2), 7゜3 (s, IH, Hl4): 8 ．． 4 (s, IH, Hl5); "CNMR (020) ppm 154.2. 2CO; 168.3.4CO; 116.2.5C; 141.1.6C; 2 5, 8.7C; 37.4.8C; 41.8 & 41.2.9C & IO C; 55.7° 11C; 30.3.12c; 131.0.13c, 12 0.6.14C: 138.1.15C; 178.3.16C; 171.9 ．． 17C; 144.9.18C; 98.9.19c: 159.3.20C: 168.2.21C; 88.3.1'X; 88.9.1'C; 40.8 ゜2'X: 42.3.2'C; 77.8.3'X (Jc, = 5.1 Hz) ; 73.3.3'C; 88.7 (J,, = 6.5 Hz), 4'X; 8 8.1.4'C (Je, = 8.9 Hz); 64.0.5'X; 67.8.5 'C (Jc, = 5.05 Hz).

chill]-5-[3-[2-[[2-[[(2,2-dimethylethoxy)carbo Nyl]-amino]-3-[[1-(2,2-di-methylethoxy)carbonyl] -1H-imidazol-4-yl]-1-oxobrobyl]amino]ethyl]a mino-3-oxobropyl]-2'-deoxy-uridine-3'-0-(N, N-diisopropyl-amino-q-cyanoethoxy)phosphine (IIA) preparation Chloro-N,N-diisopropylamino-q-cyanoethoxyphosphine (0, 158 g, 0.81 mmol) was weighed into an H-type Schlenk flask, and Dissolved in tril (10 ml). Diisopropylethylamide (0,196 mL 1.52 mmol) was added to the reaction vessel and the mixture was stirred at room temperature for 20 minutes. Nu Cleoside (7A) (0.75g, 0.furomillimole) dissolved in acetonitrile and added to the phosphorylation mixture and stirred for 30 minutes. Then flip the mixture to the other side of the H tube to remove the amide hydrochloride. solids Wash with setonitrile (2XIOml), concentrate the combined M e CN solution, and A lath-like substance was obtained. Next, the glassy substance was transferred to a silica gel chromatotron (2000 Chromatography was performed on M). Phosphoramidite (IIA) (0,72 1 g, 0.61 mmol, 80%) is CHt C1! :EtOAc :E Eluted at taN=4.5:4.5:l. ”PNMR(CD3CN)p pm149゜4゜2 S (diastereoisomer). FABMS, m/zl19 4. (M+2Li);1188. (M+Lf);1094. (M+2Li-H- Boc); 994, (M+3Li-H-Boc); accurate mass measurement 1188. 5789, Cs. Hl. Calculated value for Ns O14P L j is 1188.5 722° XI) Preparation of T (12A) Phosphoramidite (IIA) was used on the Pharmacia Gene Assembler. dinucleoside 5'-0-DMT-X' pT was synthesized and completely deprotected. The product was protected and purified on an Artic Econosil CI8 preparative RPHPLC column. ( 12A) (in 250D units) is the same line with a flow rate of 1.5ml/min. On an analytical column using a ternary gradient end it was 14.5 minutes. 2HNM R(D, 0) d 6.3 (t, IH, 1'X); 6.4 (t, IH , 1'T); 2.55 (m, 2H, 2'X); 2.4 (m, 2H, 2 'T); 4.65 (m, 18.3'T); 4.85 (m, IH, 3'X ): 4.2 (m, IH, 4'X); 4.0 (m, IH.

4'H); 3.85 (m, 2H, 5'X); 4.2 (m, 2H, 5 °T): 7.75 (S.

IH, H6); 7.75 (s, IH, H21); 2.4 (2t’s, 4H, H7 &H8); 3.3 (m, 4H, H9 &HIO); 4. 35 (m, IH, Hll); 3.1 (m, IH, Hl2); 7.2 (s, IH, Hl4); 8.2 (s, IH, Hl5); 1.95 (s , 3H, T-CHs).

Phosphoramidite (14A) was used on the Pharmacia Gene Assembler. , 11-mer 5'-Ho-TpA' p'r' p(:' p7pTpC' pTpXpA'pC'-0-S-3' was synthesized (10 mmol scale).

Oligomer on solid support was dissolved in 10% cF, C0OH was dissolved in CH*CI* The mixture was washed with water for 10 minutes to perform detritylation and remove the Boc group. Deprotection was completed with aqueous NHs (0,88d) and the product was converted to nucleogen-DEAE6. Purified on a 0-7 preparative HPLC column.

Oligo 7-(13A) (900 D units) was added to the same solvent group at a flow rate of 1.5 ml/min. Using Radiend, it was eluted from the ion exchange column for analysis at 23.5 minutes.

0.00 2.00 4.00 6.00 8.00FIG, IA FIG. 18 FIG. 2 FIG, 3A FIG. 3B FIG. 4A FIG. 4B FIG, 5A FIG.5B f FIG. 7A f FIG. 7B LANE 1 2 3 4 5 6 7 8 FIG, 9 FIG. 17A Summary book Selected sequence-directed hydrolysis of RNA under physiologically suitable conditions A conjugate comprising a metal complex covalently bonded to an oligodeoxynucleotide as a cleavage agent. Describe using union. The oligodeoxynucleotide portion of this drug is Molecular recognition using Watson-Crick base pairing is performed on the target RNA sequence to be decoded. selected as follows. Metals used to form metal complexes useful in the present invention A method for measuring the effects of RNA hydrolysis of ligands and ligands is described. as a cleavage agent Contains two or more imidazole groups covalently bonded to the oligodeoxynucleotide of selected sequence-directed cleavage of RNA using a conjugate consisting of comprising one or more imidazole groups covalently attached to a oligodeoxynucleotide. The ribodeoxynucleotide moiety is a nucleotide relative to the sequence of the target RNA to be cleaved. selected for molecular recognition through single-click base pairing.

Copy and translation of amendment) Submission form (formerly 1992 1 of mPFt*fR1841) February 14th Nikko

Claims (1)

[Claims] 1. A method for hydrolytically cleaving RNA under physiologically suitable conditions with a compound selected from the group consisting of nucleosides, nucleotides and oligodeoxynucleotides bound to metal complexes effective for RNA hydrolysis. 2. The above-mentioned metal complexes have been shown to be involved in the addition of RNA as shown by Applicants' HPLC analysis method. 2. A method according to claim 1, which exhibits water splitting. 3. 3. The method of claim 2, wherein said metal complex is attached to said compound at any position where chemical derivatization is possible. 4. Copper, zinc, cobalt, nickel, palladium, lead, iridium, manganese, iron, molybdenum, vanadium, ruthenium, bismuth, magnesium, uranium 3. The method of claim 2, wherein the lanthanide metal is selected from the group consisting of rhodium, rhodium, and lanthanide metals. 5. Claims wherein said metal is selected from the group consisting of copper, zinc and cobalt. The method described in section 2. 6. The ligand of the metal complex is 2,2':6',2''-pyridine;2,2'-bipyridine;7-(N-methyl)-2,12-dimethyl-3,7,11,17 -tetraazabicyclo[11,3,1]heptadeca-1(17),2,11,13,15-pentaene; 2,12-dimethyl-3,7,11,17-tetraazabicyclo[11.3.1 ]Heptadeca-1(17),2,11,13,15-pentaene;2,10-dimethyl-3,6,9,12-tetraazabicyclo[9.3.1]pentadeca-1(15),2 ,10,12,15-pentaene; N-phosphonomethyliminodiacetic acid and 4,4'-dimethyl-2,2'-bipyridi 3. A method according to claim 2, wherein the method is selected from the group of compounds consisting of: 7. The above complexes include 2,2':6',2''-terpyridine and copper(II); 2,2'-bipyridine and copper(II); 4,4'-dimethyl-2,2'-bipyridine and Copper (II); 7-(N-methyl)-2,12-dimethyl-3,7,11,17-tetraazabicyclo[11.3.1]heptadeca-1(17),2,11,13, 15-Pentaene and zinc (II); 2,12-dimethyl-3,7,11,17-tetraazabicyclo[11.3.1]heptadeca-1(17),2,11,1 3,15-pentaene and 2,10-dimethyl-3,6,9,12-tetraazabicyclo[9.3.1]pentadeca-1(15),2,10,12,15-pentaene and copper ( II), wherein said complex is selected from the group of complexes formed by reacting with chloride, hydroxide, water, bromide, iodide. having auxiliary ligands selected from the group consisting of mono- and bidentate ligands, perchlorates, nitrates, sulfates, phosphines, phosphites, and other mono- and bidentate ligands. 2. The method according to claim 2, wherein: 8. A compound comprising a metal complex effective to hydrolyze covalently attached RNA at any position capable of chemical derivatization into a nucleoside, nucleotide or oligodeoxynucleotide. 9. Showing the hydrolysis of RNA as shown by Applicants' HPLC analysis method, Claim 8, wherein the metal complex is a nucleoside or a nucleotide. Compounds listed. 10. The complex exhibits sequence-directed hydrolysis of RNA, and the complex forms oligodeoxynucleolyses. 9. A compound according to claim 8, which is bonded to tide. 11. The metal complexes are 2,2':6',2''-terpyridine and copper(II); 2,2'-bipyridine and copper(II); 4,4'-dimethyl-2,2'-bipyridine. 7-(N-methyl)-2,12-dimethyl-3,7,11,1 7-tetraazabicyclo[11.3.1]heptadeca-1(17),2,11 , 13,15-pentaene and zinc (II); 2,12-dimethyl-3,7,11, 17-tetraazabicyclo[11.3.1]heptadeca-1(17),2,11,13,15 -pentaene and zinc (II); and 2,10-dimethyl-3,6,9,12-tetraazabicyclo[9.3.1]pentadeca-1(15),2,10,12,15-pentaene Formed by reacting with copper(II) complexes selected from the group consisting of chlorides, hydroxides, water, bromides, iodine, urides, perchlorates, nitrates, sulfates, phosphines, sulfites, and other molecules 9. A compound according to claim 8, which may have an auxiliary ligand selected from the group consisting of no- and bidentate. 12. The metal complex may be a 2'-deoxy-uridine nucleoside or a nucleoside. C-5 position of uracil of otide, 2'-deoxy-cytidine nucleoside or C-4 or C-5 position of cytosine of cleotide, 2'-deoxy-adenosine N-6 position or 2'-deoxy group of adenine in a creoside or nucleotide Anosine nucleoside or nucleotide N-2 or 0-6 position of guanine 9. A compound according to claim 8, which is outside the scope of claim 8. 13. The metal complexes are 2,2':6',2''-terpyridine and copper(II); 2,2'-bipyridine and copper(II); 4,4'-dimethyl-2,2'-bipyridine. 7-(N-methyl)-2,12-dimethyl-3,7,11,1 7-tetraazabicyclo[11.3.1]heptadeca-1(17),2,11 , 13,15-pentaene and zinc (II); 2,12-dimethyl-3,7,11, 17-tetraazabicyclo[11.3.1]heptadeca-1(17),2,11,13,15 -pentaene and zinc (II); and 2,10-dimethyl-3,6,9,12-tetraazabicyclo[9.3.1]pentadeca-1(15),2,10,12,15-pentaene Formed by reacting with copper(II) complexes selected from the group consisting of chlorides, hydroxides, water, bromides, iodine, Uride, perchlorate, nitrate, sulfate, phosphine, phosphite, and other molecules having an auxiliary ligand selected from the group consisting of no- and bidentate ligands. 13. A compound according to claim 12, which is capable of 14. The metal in said metal complex is selected from the group consisting of copper, zinc and cobalt. 9. A compound according to claim 8, wherein 15. 9. A compound according to claim 8, wherein the ligand in said metal complex is selected from the group consisting of 2,2'-bipyridine and substituted 2,2'-bipyridine. 16. Claim 8, wherein the ligand in said metal complex is selected from the group consisting of 2,2':6',2''-terpyridine and substituted 2,2':6',2''-terpyridine. Compounds described in Section. 17.5-[3-[[2-[[4-[4'-methyl[2,2'-bipyridine]-4-yl]-1-oxobutyl]-amino]ethyl]amino]-3-oxopro Complex of pyl]-2'-deoxy-uridine and copper(II). 18.3'-[4-[4'-methyl[2,2'-bipyridin]-4-yl]buty Complex of ammonium salt]-phosphate]-2'-deoxy-thymidine and copper(II). 19.5'-[4-[4'-methyl(2,2'-bipyridin)-4-yl]buty complex of triethylammonium salt of 2'-deoxy-thymidine and copper (II). 20. A method for covalently bonding a metal complex effective in hydrolyzing RNA to a nucleoside, nucleotide or oligodeoxynucleotide, the method comprising: the nucleotide or oligodeoxynucleotide and the metal complex as described above. reacting the nucleoside, nucleotide or oligodeoxynucleotide under conditions in which the genus complex covalently bonds to said nucleoside, nucleotide or oligodeoxynucleotide at any position where chemical derivatization is possible. 21. When the nucleoside, nucleotide, or oligodeoxynucleotide is reacted with an organic ligand to form a complex with a metal ion effective in hydrolyzing RNA, the organic ligand is covalently bonded to the nucleoside, nucleotide, or oligodeoxynucleotide. After that, the resulting compound is added to RNA. The metal ions are reacted with the metal ions effective for water splitting, and the metal ions are reacted with the organic ligand. 21. The method according to claim 20, characterized in that the method is coupled to a code. 22. Said metal ion is first reacted with said ligand to form a complex, which is then reacted with said nucleoside, nucleotide or oligodeoxynucleotide. 21. The method of claim 20, wherein the complex is covalently bonded to the complex at any position where chemical derivatization is possible. 23. An improved method of hydrolyzing RNA under physiologically suitable conditions, comprising contacting said RNA with a metal complex covalently attached to an oligodeoxynucleotide to provide sequence-directed hydrolysis. . 24. 24. The method of claim 23, wherein said metal complex exhibits hydrolysis of RNA as indicated by Applicants' HPLC analysis method. 25. 24. The method of claim 23, wherein said metal complex is attached to said oligodeoxynucleotide at any position where chemical derivatization is possible. 26. If the above metals are copper, zinc, cobalt, nickel, palladium, lead, or um, manganese, iron, molybdenum, vanadium, ruthenium, bismuth, magnetite 24. The method of claim 23, wherein the metal is selected from the group consisting of sium, uranium, rhodium and lanthanide metals. 27. 24. The method of claim 23, wherein said metal is selected from the group consisting of copper, zinc and cobalt. 28. The recant of the metal complex is 2,2':6',2''-terpyridine; 2,2'-bipyridine; 7-(N-methyl)-2,12-dimethyl-3,7,1 1, 17-tetraazabicyclo[11.3.1]heptadeca-1(17),2, 11,13,15-pentaene; 2,12-dimethyl-3,7,11,17-te Traazabicyclo[11.3.1]heptadeca-1(17),2,11,13,15-pentaene:2,10-dimethyl-3,6,9,12-tetraazabicyclo b[9.3.1]pentadeca-1(15),2,10,12,15-pentaene; N-phosphonomethyliminodiacetic acid and 4,4'-dimethyl-2,2'-bipylene 24. The method of claim 23, wherein the method is selected from the group of compounds consisting of lysine. 29. The metal complexes are 2,2':6',2''-terpyridine and copper(II); 2,2'-bipyridine and copper(II); 7-(N-methyl)-2,12-dimethyl Ru-3,7,11,17-tetraazabicyclo[11.3.1]heptadeca-1 (17), 2,11,13,15-penquene and zinc(II); thyl-3,7,11,17-tetraazabicyclo[11.3.1]heptadeca-1(17),2,11,13,15-pentaene and zinc(II); and 2,1 0-dimethyl- 3,6,9,12-tetraazabicyclo[9.3.1]pentade Reacting car-1(15),2,10,12,15-pentaene with copper(II) selected from the group of complexes formed by 24. The method of claim 23, which may have an auxiliary ligand selected from the group consisting of phosphate, and other mono- and bidentate ligands. 30. RNA is converted into a nucleoside with two or more imidazole groups bonded to it. opened with a compound selected from the group consisting of otides and oligodeoxynucleotides. How to tear. 31. The imidazole group may be attached to the compound at any position where chemical derivatization is possible, as long as the essential properties of the imidazole remain intact, so that they are acidic in the Lewis or Prensted definition of the term. 31. A method according to claim 30, wherein the base is capable of acting as a base or both. 32. RNA is converted into a nucleoside, a nucleoside, each linked with one or more imidazole. A method of cleavage using a combination of two compounds selected from the group consisting of nucleotides and oligodeoxynucleotides. 33. The imidazole group may be attached to the compound at any position where chemical derivatization is possible, as long as the essential properties of the imidazole remain intact, so that they are acidic in the Lewis or Frenstead definition of the term. 33. A method according to claim 32, wherein the base is capable of acting as a base or both. 34. Nucleosides, nucleotides and oligodeos having at least one imidazole group attached thereto in the presence of a solution containing at least one imidazole group. A method of cleaving RNA with a compound selected from the group consisting of xynucleotides. 35. The imidazole group may be attached to the compound at any position where chemical derivatization is possible, as long as the essential properties of the imidazole remain intact, so that they are acidic in the Lewis or Frenstead definition of the term. 33. A method according to claim 32, wherein the base is capable of acting as a base or both. 36. imidazole Chemical derivatization is possible as long as the essential properties of A nucleoside, nucleotide or oligodeoxynucleotide at any position where A compound comprising two or more imidazole groups effective to cleave RNA covalently bound to a compound that can act as an acid, a base, or both within the Lewis or Frenstead definition of the term. Compounds that include: 37. shows sequence-directed cleavage of RNA, and the imidazole group 37. A compound according to claim 36, which is attached to a cleotide. 38. The imidazole groups are juxtaposed in a suitable manner to promote cleavage of the RNA. 37. A compound according to claim 36, wherein the compound is 39.5-[3-[[2-[[2-[[2-amino]-1-oxo-3-[1H-imidazol-4-yl]propyl]amino]-1-oxo-3-[1H −i midazol-4-yl]propyl]amino]ethyl]amino]-3-oxopro pill]-2'-deoxyuridine compound. 40. Chemical derivatization is possible as long as the essential properties of imidazole remain intact. two or more nucleosides, nucleotides or oligodeoxynucleotides at any position where one or more imitations effective to cleave RNA covalently bound to the oxynucleotide; A combination of compounds comprising a dazole group, wherein they are Lewis or fluorine groups. A compound that acts as an acid, a base, or both in the Lönsted definition of the term. A combination that includes enabling and. 41. The imidazole groups are juxtaposed in a suitable manner to promote cleavage of the RNA. 41. The combination according to claim 40, wherein: 42. Two or more imidazole groups effective for RNA cleavage are added to a nucleoside or nucleoside. This is a method of covalently bonding to otides or oligodeoxynucleotides. When a cleoside, nucleotide or oligodeoxynucleotide is combined with an imidazole group, the reactivity of imidazole allows further chemical reactions to occur. The nucleic acid can be used at any position where chemical derivatization is possible, as long as this is not prevented. A method of reacting under conditions to covalently bond osides, nucleotides, or oligodeoxynucleotides. 43. In a method of cleaving RNA under physiologically suitable conditions, said RNA is cleaved with an oligodeoxynucleotide or two or more oligodeoxynucleotides having one or more imidazole groups covalently attached to provide sequence-directed cleavage. deoxynucleo a compound selected from the group consisting of two or more imidazole groups covalently bonded to An improved method involving contact with a compound. 44. In a method of cleaving RNA under physiologically suitable conditions, the RNA is co-coated with an oligodeoxynucleotide in the presence of a solution containing an imidazole group. contact with a compound selected from the group consisting of one or more imidazole groups bonded; Improved method, including touching. 45. Said imidazole group can be added to said oligodeoxynucleotide at any position where chemical derivatization is possible as long as the essential properties of imidazole are not impaired. as claimed in claim 43, covalently bonded to the otide, enabling them to act as an acid, a base or both in the Lewis or Frenstead definition of the term. How to put it on.