AU643542B2 - RNA hydrolysis/cleavage - Google Patents

Description

OPI DATE 07/01/92 INTERNI AOJP DATE 13/02/92 INTERN/ APPLN- ID 80679 91 PCT NUMBER PCT/US91/03880 TREATY (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 91/19730 CO7H 23/00, A61K 31/70 Al (43) International Publication Date: 26 December 1991 (26.12.91) (21) International Application Number: PCT/US91/03880 (74) Agent: BOLOING, James, Clifton; Monsanto Company, 800 North Lindbergh Boulevard, St. Louis, MO 63167 (22) International Filing Date: 3 June 1991 (03.06.91) (US).

Priority data: (81) Designated States: AT (European patent), AU, BE (Euro- 550,001 14 June 1990 (14.06.90) US pean patent), CA, CH (European patent), DE (Euro- 609,497 5 November 1990 (05.11.90) US pean patent), DK (European patent), ES (European patent), FR (European patent), GB (European patent), GR (European patent), IT (European patent), JP, LU (Euro- (71) Applicant: MONSANTO COMPANY [US/US]; 800 pean patent), NL (European patent), SE (European pa- North Lindbergh Boulevard, St. Louis, MO 63167 tent), SU.

(72) Inventors: BASHKIN, James, Keane 7739 Stanford Avenue, University City, MO 63130 MODAK, Anil, Published Shrikrishna 1193F Schulte Hill Drive, Maryland With international search report.

Heights, MO 63043 STERN, Michael, Keith 1075 Before the expiration of the time limit for amending the Wilson Avenue, St. Louis, MO 63130 claims and to be republished in the event of the receipt of amendments.

643542 (54) Title: RNA HYDROLYSIS/CLEAVAGE (57) Abstract The selecto directed hydrolysis of RNA under physiologically relevant conditions is described using conjugates comprising mal co,.',,cxes covalently linked to oligodeoxynucleotides as hydrolysis agents. The oligodeoxynucleotide portions of the agents are selected to provide molecular recognition via the Watson Crick base pairing to the target RNA sequence to be hydrolyzed. A method is described for determining the RNA hydrolysis effectiveness of metals and ligands used to form the metal complexes useful in this invention. Also the selected sequence-directed cleavage of RNA is described using conjugates co:mprising two or more imidazole groups covalently linked to oligodeoxynucleotides as cleavage agents and combinations of conjugates comprising one or more imidazole groups covalently linked to two or more oligodeoxynucleotides. The oligodeoxynucleotide portions of the agents are selected to provide molecular recognition via the Watson Crick base pairing to the target RNA sequence to be cleaved.

WO 91/19730 V~~O 9/I973OPCY/US9I /03880 RUA HYDROLYSI Si CLEAVAGE FIELD OE' INVEION This invention relates to sequence-directed RNA hydrolysis under physiologically relevant conditions and particularly to metal complexes covalently linked to oligodeoxynucleotides as sequence-directed RNA hydrolysis age~nts and to RNA cl~ivage generally and particularly to two or more imidazoles covalenttly linked to oligodeoxynucleotides as sequence-directed RNA cleavage agents.

BACEGROUN OF THE INVNTI Recently hydrolysis phosphate esters has received extensive invest igak; on as reported in the art due to tho relevance of this chemistry to biological systems, and specifically transition metal complexes have been examined as phosphate ester hydrolysis catalysts in order to model the reactions catalyzed by the ATPase and phosphatase classes of enzymes and also particularly as it relates to the manipulation of the phosphodiester backbone of ribonucleic acids (RNA).

Such reported studies have generally employed activated p-nitrophenyl phosphiate esters or phosphate anhydrides (ATP) ais substrates D. Cornelius, Inorg. Chem. 1980, 19, 1286-1290; P. R. Norman et al, J. Am. Chen. Soc.

1982, 104, 2356-2361 and F. Tafesse et al, Inorgr. Chem.

1955, 24, 2593-2594). -It ham been reported that tetrazine complexts of Ca#01II) are capable of promoting the hydrolysis of adenosine 31,5-monophosphate (cAMP) Chin et al, Can. Chem. 1987, 65, 1882-1884) and adenosine monophosphate (AMP) Chin et al, J. Ax.

Chem. Soc. 1989, 111, 4103-4105). Also, it is known that xany divalent cations are capable of catalyzing the hydrolysis of RNA J. Butzow et al. Blochehistry 1971, 10, 2016-2027 and Z. J. Butzov et al, Nature 1975, 254, 358-359). Additionally, Fin 'c ion in the presence of imidazole buffers hats been shown to, catalyze the hydrolysis of the RNA dimer 30,51-UpU at 800C. (R.

Breslow et al, Proc. Natl. Acad. Scl. 1989, 86, 1746- WO 91/19730 PCT/US91/03880 -2- 1750). The ribonuclease class of enzymes is known to hydrolyze RNA in vivo and in vitro (Blackburn et. al.

The Enzymes; Academic Press: New York, 1982; Vol Chapter 12, pp 317-433.), and the.active site of many ribonucleases contains histidine residues believed to be involved in catalysis (Richards et al. Ibid., 1971, Vol.

4, Chapter 24, pp 647-806). In order to model the reactions catalyzed by the ribonucleases, investigations into phosphate ester hydrolysis have been carried out using imidazole and imidazole derivatives as models for histidine. Such reported studies have generally employed activated p-nitrophenyl phosphate esters as the hydrolysis substrate, instead of RNA itself (Anslyn et al. J. Am. Chem. Soc. 1989, 111, 5972-5973, and Anslyn et al. Ibid. 8931-8932.). These activated esters are more easily studied than the true biological substrates for two reasons: they are more easily hydrolyzed, and since the product p-nitrophenolate anion has a strong characteristic color, the reaction may be followed by simple spectrochemical techniques. While these analogues are convenient models for biological substrates, they are not accurate models (Menger, F. M. and Ladika, J. Am. Chem. Soc. 1987, 109, 3145.) C. A. Stein et al, Cancer Research May, 1988, 48, 2659-2668 gives a detailed review on the application of antisense oligodeoxynucleotides as modulators of gene expression and concludes by proposing a more subtle and effective approach would be to attach a chemical group to the aligomer that can result in localized catalytic hydrolysis of RNA. This technique would be more specific than the use of a radical-producing group such as iron EDTA. Stein et al theorizes that a suitable RNA hydrolysis group would be an imidazole group, which is known to be involved in phosphodiester hydrolysis in the active site of ribonuclease enzymes.

The hydrolysis of RNA by imidazole buffers has been reported in the art (Breslow et. al. J. Am. Chem.

Soc. 1986, 108, 2655-2659) and the mechanism of this WO 91/19730 PCT/US91/03880 -3reaction has been studied (Anslyn et. al. J. Am. Chem.

Soc. 1989, 111, 4473-4482). Based on these studies, a bifunctional general acid-general base mechanism was proposed for the hydrolysis of RNA by imidazole.

University Patents, Inc. in PCT International Patent Application published under number WO 88/04300 on June 16, 1988 discloses RNA enzymes or ribozymes, acting as endoribonucleases, as catalyzing the cleavage of RNA molecules with a sequence specificity of cleavage greater than that of known ribonucleases and approaching that of the DNA restriction endonuclsases, thus serving as RNA sequence-specific endoribonucleases. Ribozymes are entirely or partly comprised of RNA itself, and therefore are chemically and enzymatically highly unstable relative to Applicants' DNA-based compounds.

Such instability detracts from the practical applicability of RNA hydrolysis agents. Also ribozymes presently are available anly at a high cost due to limitations of very low production volumes through molecular biology techniques.

C. B. Chen et al, J. Am. Chem. Soc. 1988, 110, 6570-6572 describes that 1,10-phenanthroline-copper(II) is effective for targeted cleavage of both RNA and DNA and thus is useful for sequence-specific cleavage of RNA. This teaching is directed to oxidative cleavage of RNA by metal complexes linked to DNA at a temperature of as opposed to the hydrolytic cleavage of RNA under physiologically relevant conditions required by Applicants' invention. The ancillary reagents, in the quantities required to drive the Chen et al oxidative degradation of RNA, are not compatible with living cells; furthermore, the 1,10-phenanthrolinecopperoligodeoxynucleotide conjugate employed is itself degraded oxidatively under the conditions of oxidative RNA cleavage (the rate of oxidative cleavage by the 1,10-phenanthrolinecopper system'is similar for both RNA and DNA).

WO 91/19730 PC/US91/03880 -4- P. G. Schultz and coworkers in a series of articles R. Corey et al, J. Am. Chem. Soc. 1988, 110, 1614-1615; R. Zuckerman et al, J. Am. Chem. Soc.

1988, 110, 6592-6594 and R. Zuckerman et al, Proc. Natl.

Acad. Sci. USA 1989, 86, 1766-1770) have described the preparation of site-selective DNA and RNA hydrolysis agents comprised of an enzyme (staphylococcal nuclease, ribonuclease S, or mutants of these parent enzymes) covalently linked to oligonucleotides. In one report R. Corey et al, Blochemistry 1989, 28, 8277-8286), the location of the linker arm and its length were varied, which resulted in changes in catalytic efficiency and site of cleavage. These nucleic acid hydrolysis agents differ from our inventions in several important aspects: the nucleic acid cleavage behavior is provided by an enzyme, not the synthetic, small molecule hydrolysis agent (or enzyme mimic) that we have disclosed; enzymes are subject to proteolytic degradation by other enzymes; staphylococcal nu-lease is dependant on added calcium for its activity; ribonuclease S is a noncovalent complex comprised of the S-protein and S-peptide derived from ribonuclease A, and this complex is subject to dissociation, which results in loss of cleavage efficiency and specificity; oligonucleotide-staphylococcal nuclease conjugates were found to cleave DNA as well as RNA, thus lacking the specificity of our agents for RNA hydrolysis alone; this high activity limits the specificity of tha enzyme-based systems developed by Schultz, because nonspecific cleavage events are common; the specificity of the enzyme-based systems was artificially increased by lowering the temperature below physiologically relevant values to 0*C).

Considerable art has been developed on cleavage of RNA utilizing enzymes and ribozymes. At present the art is void of a teaching using metal complexes which cleave RNA hydrolytically at a physiologically relevant pH and temperature as synthetic analogs for enzymes or WO' 91/19730 PCT/US91/03880 ribozymes to obtain sequence-directed hydrolysis of RNA.

Sequence-directed RNA hydrolysis and RNA cleavage are highly desirable today in order to prepare catalytic antisense oligodeoxynucleotides useful as a means for inhibiting the expression of specific genes. From several practical perspectives, the fragile, high molecular weight enzyme-oligonucleotide conjugates described by Schultz et al are at an extreme disadvantage in comparison with the low molecular weight, stable, synthetic ribonucleases of this invention. These practical points include the ability to prepare more than minute quantities; the ability to prepare high-purity material free from contaminating activities or unknown, inactive impurities; the potential to survive in vivo conditions and to avoid immunological responses; and the ability to avoid nonspecific hydrolysis of both DNA and RNA. The art is also void of a teaching using imidazole groups attached to nucleosides, nucleotides and oligodeoxynucleotides as synthetic analogues for enzymes or ribozymes for the catalytic cleavage of RNA to obtain sequence-directed cleavage of RNA. Such RNA hydrolysis and cleavage are necessary to provide a basis for catalytic antisense drug development.

STATEMENT OF THE INVENTION This invention is directed to the hydrolytic cleavage of RNA at physiologically relevant conditions.

The underlying basis of this invention is the use of metal complexes which perform as synthetic analogs for enzymes or ribozymes in the hydrolysis of RNA. This invention is also directed to the cleavage of RNA by nucleosides, nucleotideis and oligodeoxynucleotides containing two or more imidazole groups attached via an appropriate linker, to the cleavage of RNA by a combination of two oligodeoxynucleotides each containing at least one imidazole group attached via an appropriate linker, and to the cleavage of RNA by an imidazole group attached via an appropriate linker to a nucleoside, WO 91/19730 PCT/US91/03886 -6nucleotide or oligodeoxynucleotide in the presence of an imidazole group in solution. The imidazole groups perform as synthetic analogs for the active sites of enzymes or ribozymes in the cleavage of RNA. Conjugate as used herein means a compound comprised of a metal complex covalently linked to a nucleoside or nucleotide or a compound comprised of two or more imidazole groups covalently linked to a nucleoside or nucleotide, or a combination of two or more nucleosides or nucleotides each having one or more imidazole groups covalently linked thereto. Oligodeoxynucleotide conjugate as used herein means a compound comprised of a metal complex covalently linked to an oligodeoxynucleotide or to a compound comprised of one or more imidazole groups covalently linked to an oligodeoxynucleotide, or a combination of two or more oligodeoxynucleotides each having one or more imidazole groups covalently linked thereto. The term imidazole group as used herein includes imidazole and analogs of imidazole, including nitrogen-containing compounds which retain the essential properties of the imidazoles, so that they may function as either acids, bases or both, in either the Lewis (Cotton and Wilkinson, Advanced Inorganic Chemistry, 1988, Wiley, NY, p36); Orchin et al (The Vocabulary of Organic Chemistry, Wiley, 1980, p248); or Brensted (Orchin et al, The Vocabulary of Organic Chemistry, Wiley, 1980, p249) definitions of the terms.

A first aspect of this inention is directed to the discovery of metal complexes useful for promoting RNA hydrolysis. A second aspect of this invention is directed to a conjugate which is active for RNA hydrolysis comprised of a metal complex covalently linked to a nucleoside or nucleotide. A third aspect of this invention is directed to the sequence-directed hydrolytic cleavage of RNA by a metal complex covalently linked to an oligodeoxynucleotide. Another aspect of this invention is directed to the discovery of two or more imidazole groups covalently linked to nucleosides, WO 91/19730 PCT/US91/03880 -7nucleotides and oligodeoxynucleotides useful for pronoting RNA cleavage. The oligodeoxynucleotide provides molecular recognition via Watson Crick base pairing to the target RNA sequence.

Accordingly, major objects of this invention are to provide for the hydrolysis of RNA at physiologically relevant conditions and for the cleavage of RNA. Other objects of this invention include the discovery of metal complexes which are effective for the hydrolysis of RNA, the discovery that molecules containing two or more imidazole groups show great enhancement over mono-imidazole species for the cleavage of RNA, the preparation of conjugates which retain RNA cleavage behavior, the preparation of oligodeoxynucleotide conjugates effective for the sequence-directed hydrolysis of RNA under physiologically relevant conditions and the sequence-directed cleavage of RNA.

Other objects and advantages of this invention will become apparent upon further study of this disclosure and the appended claims.

DESCRIPTION OF THE DRAWINGS The structures of the compounds identified below by bold numbers in parenthesis are shown in rtAction "Schemes" 1-9.

In Figure 1 there is shown a typical example of Applicants' HPLC assay of the hydrolysis of RNA [poly(A) 12 1 8 by the metal complex, Zn-N-Me(CR). A, Time 0 hours; B, Time 18 hours.

In Figure 2 there is shown the titration of 3'- [4-[4'-methyl(2,2'-bipyridin)-4-yl]butyl-phosphate]-2'deoxy-thymidine ammonium salt with CuC12 forming 3'- [4-[4'-methyl(2,2'-bipyridin)-4-yl]butyl-phosphate]-2'deoxy-thymidine ammonium salt copper(II) This Figure depicts Applicants' Example IV and demonstrates the formation of a metal complex nucleotide conjugate in accordance with Applicants' ivWention.

In Figure 3 there is shown the hydrolytic cleavage of RNA (poly(A) 12 1 by compound This 391/19730 PCT/US91/03880 -8- Figure depicts Applicants' Example V and demonstrates that a metal complex linked to the 3' position of a nucleotide is capable of hydrolyzing RNA. A, Control reaction, time 48 hours; B, Reaction of with RNA, time 48 hours.

In Figure 4 there is shown the hydrolytic cleavage of RNA [poly(A) 1218 by 5'-E4-[4'-methyl(2,2'bipyridin)-4-yl]butyl-phosphate]-2'-deoxy-thymidine triethylammonium salt copper(II) This Figure depicts Applicants' Example VIII and demonstrates that a metal complex linked to the 5' position of a nucleotide is capable of hydrolyzing RNA. A, Control reaction, time 48 hours; B, Reaction of (12) with RNA, time 48 hours.

In Figure 5 is shown the hydrolytic cleavage of RNA [poly(A) 1 .is by bipyridin]-4-yl]-1-oxobutyl]amino]ethyl]amino]-3oxopropyl]-2'-deoxy-uridine copper(II) This Figure depicts Applicants' Example XI and demonstrates that a metal complex linked to the base portion of a nucleoside is capable of hydrolyzing RNA. A, Control reaction, time 48 hours; b, Reaction of (17) with RNA, time 48 hours.

In Figure 6 there is shown the reaction of the Cu(bpy) 2 complex with both DNA [poly(dA) 121 8 and RNA [poly(A) 1 2 1 8 This Figure depicts Applicants' Example II and demonstrates that the observed cleavage of RNA by the Cu(bpy)2 complex is hydrolytic in nature and not oxidative. A, Control reaction with DNA, time 18 hours; B, Reaction of the Cu(bpy) 2 with DNA, time 18 hours; C, Control reaction with RNA, time 18 hours; D, Reaction of the Cu(bpy) 2 complex with RNA, time 18 hours.

In Figure 7 there is shown the densitometry results of polyacrylamide gel electrophoresis analysis of the sequence-directed hydrdlysis of tRNA 7y by the oligodeoxynucleotide-Cu(bpy) 2 conjugate This Figure depicts Applicants' Example XV showing WO 91/19730 WO 919730PT/US9I /03880 -9- Densitometry scans of the po3.yacrylamide gel of the reaction of (32) with tRNA Tyr af ter 17 hours under the conditions described in Example XV and of the control reaction. A, Cleavage Reaction: 1.29AM tRNA Tyr, 6. 4 M 12.9AM Cu(trpy) 227MM Cu(SO,), 5OmM NaCi and Tris buffer pH 7.8; Control Reaction: 1.29 M tRNATyr 12.9 M Cu(trpy) 227 H Cu(SO.), 50mM NaCi and mM Tris buffer pH 7.8; B, Cleavage Reaction: 1.29M tRNA Tyr, 6.4 M 12.9MM Cu(trpy )2 2271AM Cu(S0 4 NaCi and 50mM Tris buffer pH 7.8; Control Reaction: 1.2MM tRNA

T

yr, 12.9gM Cu(trpy)2, 227MM Cu(S0 4 and 6.4M l4mer-oligodeoxynucleotide 5' -HO-TGACGGCAGATTTA-OH-3'.

In Figure 8 is shown t~he structure and identity of compounds known to Applicants which do not show RNA hydrolysis accordirl to this invention.

in Figure 9 there is shown the autoradiogrr~ph which depictz the cleavage of 32P labeled RNA by compound (SA) and that compound is ineffective at cleaving RNA. Lane RNA control t-2 hours; Lane #2 througla G, Reaction of RNA with 1 rM Lane 12, t-0 hours; Lane t-0.5 hours; Lane f4, t-1 hour; Lane hours; Lane t-2.0 hours; Lane #7 and reaction of 2mM (6A) with RNA; Lane t-0 hours; Lane t-2 hours.

In Figure 10~ there is shown a schematic view of the use of two oligodeoxynucleotide conjugates, labeled antisense Probe 1 and Probe 2, juxtaposed in a manner to enhance cleavage of RNA.

Scheme I. depicts the synthesis of compox, nd as described in Applicants# Exampl.e III.

Scheme 2 depicts the synthesis of compound (13-) as described in Applicants' Example VI.

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

Scheme 4 depicts the synthiesis of compounds and (23) as described in Applipants' Example XII.

Scheme 5 depicts the synthesis of compound as descr~ibed in Applicants' Example XIV.

WO 91/19730 9PCT/US91/0388(i Scheme 6 depicts the sequence-directed cleavage of tRNATyr by compound (32) as described in Applicants' Example XV.

Scheme 7 depicts the synthesis of compound as described in Applicants' Example XVI.

Scheme 8 depicts the sequence-directed cleavage of RNA by compound (5A) as described in Applicants' Example XVII.

Scheme 9 depicts the synthesis of compound (12A) as described in Applicants' nxample XVIII.

SUMMARY OF THE INVENTION The hydrolytically effective oligodeoxynucleotide conjugates of this invention are comprised of a desired organic molecule, herein eftorre to as the ligand, a metal ion, which imparts the hydrolytic activity, and a desired oligodeoxynucleotide. The effective conjugates of this invention are comprised of two or more imidazole groups which impart the RNA cleavage activity, and one or more desired nuclaosides, nucleotides or oligodeoxynucleotides.

In one aspect Applicants' invention is based on metal complexes which are effective for RNA hydrolysis, and the preparation of such metal complexes covalently linked to nucleosides, nuicleotides and oligodeoxynucleotides. The metal complexes covalently linked to the nucleosides, nucleotides and oligodeoxynucleotides distinguishes Applicants' invention from the speculation of the C. A. Stein et al and the teaching of the P. G.

Schultz et al references described above. Certain compounds of the type proposed by Stein et al, comprised of imidazole attached to nucleosides and oligodeoxynucleotides, were prepared and found not to be effective for RNA hydrolysis under the criteria of Applicants' invention. Such compounds and their lack of RNA hydrolysis activity are shown below in Table II as compounds (25) and Agents as used herein means Applicants' synthetic RNA hydrolysis compounds comprising a metal and a WO 91/19730 PCT/US1/03880 -11ligand, or metal complex, covalently linked to an oligodeoxynucleotide and conjugates and oligodeoxyconjugates as defined herein. The oligodeoxynucleotide provides sequence-directed recognition of RNA targets under physiologically relevant conditions. The agents of this invention are effectively artificial enzymes which mimic natural ribonucleases and ribozymes. These agents possess several advantages over ribonucleases and ribozymes in applications where sequence-directed RNA hydrolysis is desired. Such advantages include (1) enhanced specificity over ribonucleases, increased chemical stability over ribozymes, ease of production and isolation by standard chemical techniques, the ability to design sequence specificity towards any targeted RNA strand, low molecular weight, drug delivery and ability to control hydrolytic activ-it by altering the nature of the metal complex. These are important aspects of Applicants' invention which permits the practical application of sequence-directed RNA hydrolysis and cleavage. Tn.se aJpects of Applicants' invention provide considerable novelty and advantages over the prior art teachings, such as the University Patents, Inc. PCT Patent Application and the P. G. Schultz et al and C. B. Chen et al references, described above.

The nucleic acid hydrolysis compounds of Schultz et al differ from Applicants' invention in several import aspects. The nucleic acid cleavage behavior taught by Schultz et al is provided by an enzyme, not the synthetic small molecule hydrolysis agents (artificial enzymes) used by Applicants. The en ymes are subject to proteolytic degradation by other enzymes.

Staphylococcal nuclease is dependant on added calcium for its activity. Ribonuclease S is a noncovalent complex comprised of the S-protein and S-peptide derived from ribonuclease A. This complex is subject to dissociation, which results in loss of cleavage 1091/19730 PCI/US91/O3886 -12efficiency and specificity. Oligodeoxynucleotidestaphylococcal nuclease conjugates were shown to cleave DNA as well as RNA; thus, they lack the specificity of Applicants' agents for RNA hydrolysis and cleavage alone. T~is high activity limits the specificity of the enzyme-based systems developed by Schultz et al because nonspecific cleavage events are common. The specificity of these eazyme-based systems was artificially increased by lowering the temperature helow physiologically relevant values to O0C).

From several practical perspectives, the fragile, high molecular weight enzyme-oligodeoxynucleotide conjugates described by Schultz et al are at an extreme disadvantage in comparison with Applicants' low molecular weight, stable, synthetic ribonuclease analogs.

Several practical points of Applicants' invention include the ability to prepare more than minute quantities of the agents; the ability to prepare highpurity material free from contaminating activities or unknown inactive impurities; the potential to survive in vivo conditions and to avoid immunological responses and the ability to avoid nonspecific hydrolysis of both DNA and RNA.

Applicants' use of hydrolysis and use of two or more imidazole groups as the chemical reaction that cleaves RNA provides several advantages over the prior art, such as the non-selective oxidative cleavage of both RNA and DNA taught by Chen et al. Applicants' hydrolysis agents are active at pH 7 which is consistent with the conditions inside living cells. Since DNA is chemically hydrolyzed at a considerably slower rate than RNA, the sequence-directed RNA hydrolysis using Applicants' cligodeoxynucleotide conjugates will not cleave their own oligodeoxy- nucleotide components at an appreciable rate. See Applicants' Example II below.

The term oligodeoxynucleotides used herein includes oligodeoxynucleotides and oligodeoxynucleotide WO91/19730 IPCFUS91/03880 -13analogs that are.effective at molecular recognition by, for example, Watson-Crick or Hoogsteen base-pairing.

Examples of such oligodeoxynucleotide analogs include those with nonionic internucleotide linkages such as alkylphosphotriesters, alkylphosphonates and alkylphosphoramidates (as described by P. S. Miller, Oligodeoxynucleotides Antisense Inhibitors of Gene Expression, J. S. Cohen, Ed. CRC Press, Boca Raton, Florida, 198 Chapter 4 and references therein) and compounds wit sulfur-containing internuclentide linkages such as phosphorothioates and phosphorodithioates (as described by C. A. Stein et al, ibid, Chapter 5 and references therein), and alphaoligodeoxynucleotides (as described by B. Rayner et al, ibid, Chapter 6 and references therein). Other oligodeoxynucleotide analogs which may be suitable include those with internucleotide linkages such as carbonate, acetate, carbamste, dialkyl and diarylsilyl groups.

MatAl compounds applicable to this invention are those metal complex conjugates and oligodeoxynucleotide conjugates which are soluble in water at a neutral pH and are functionally effective for the hydrolytic cleavage of RNA under physiologically relevant conditions. In thk ir active forms, the metal complexes which hydrolyze RNA may contain hydroxyl or aquo ligands or both. These active forms may be derived in a standard fashion from complexes which contain ancillary ligands such as, chloride, bromide, iodide, perchlorate, nitrate, sulfate, phosphines, phosphites and other standard mono- and bidentate ligands. The metal in the metal complexes may be any metal which is effective in hydrolyzing RNA. Typical metals include copper, zinc, cobalt, nickel, palladium, lead, iridium, manganese, iron, molybdenum, vanadium, ruthenium, bismuth, magnesium, rhodium, uranium ahd the Lanthanide metals.

In order to provide the art a method of determining the applicable agents, such as metal WO 91/19730 PCT/US91/03886 -14complexes and conjugates of this invention, Applicants' have developed an assay for monitoring the hydrolysis of RNA under physiologically relevant conditions (7.1 pH and 37*C).

In another aspect Applicants' invention is based on the use of two or more imidazole groups for RNA cleavage, and the preparation of conjugates containing such imidazole groups covxlently linked to nucleosides, nucleotides and oligodeoxynucleotides.

Certain compounds of the type proposed by Stein et al, comprised of a single imidazole attached to nucleosides and oligodeoxynucleotides, were prepared and found not to be effective for RNA cleavage under the criteria of Applicants' invention.

APPLICANTS' ASSAY FOR HYDROLYSIS OF RNA A mixture of adenylic acid oligomers 12 to 18 nucleptides in length [poly(A),2.1g] is used as the assay substrate. Ion exchange HPLC is used to resolve the individual cleavage products from the substrate fragments. A compound is determined to be active if it shows hydrolytic degradation of the substrate, as illustrated in Figure. 1, to an extent greater than that which is observed for a control reaction run under identical conditions in the absence of a cleavage agent.

The extent of reaction is determined from the ratio of the integration of substrate paak at time 0 hour and time 18 hours.

HPLC analysis is performed with a Waters 600 multisolvent delivery system and a 490 programmable multiwavelength detector. Data is acquired on a NEC APC IV Advanced Personal Computer using Waters Maxima 820 software. With this system it is possible to determine the area under all the substrate peaks. Extensive standard precautions need to be taken to avoid RNase contamination: all buffers are made with diethylpyrocarbonate treated water vol./vol.) and the reactions are run in sterilized polypropylene tubes.

Typical stock solutions of RNA [poly(A),2.18] having an WO 91/19730 PCT/US91/03880 adenosine concentration of 761#M are prepared by dissolving 10 units of the RNA in 20mM HEPES buffer pH 7.1. HPLC analyses are run on a 7gM Nucleogen DEAE 7 with an elution gradient of 0-15 min. 25% B, 15-45 min. 60% B, 45-60 min. 100% B; using Solvent A

KH

2

PO

4 20% acetonitrile, pH 5.5; and Solvent B Solvent A 1M KC1.

The combination of agents applicable and useful in this invention are those which functionally promote RNA hydrolysis as determined by this assay. The above described assay is not to be considered a limitation on Applicants' invention. It is to be understood that other assays can be developed and used to determine the effectiveness of agents for the hydrolysis of RNA in accordance with this invention.

A further check for the effectiveness of the metal complexes for hydrolyzing RNA is the formation of a conjugate. In forming such conjugates, the selected ligand may first be covalently linked to the desired nucleoside or nucleotide and then the selected metal ion attached to the ligand. Alternatively, the intact selected metal complex may be covalently linked to the nucleoside or nucleotide. The ligand or intact metal complex may be covalently linked to the nucleoside or nucleotide at any location. The details of forming specific conjugates are fully described in Applicants' Examples.

Likewise, in preparing the oligodeoxynucleotide conjugates of this invention, the selected ligand may first be covalently linked to the desired oligodeoxynucleotide and then the selected metal ion attached to the ligand. Alternatively, the intact selected metal conplex may be covalently linked to the oligodeoxynucleotide. The ligand or metal complex can be covalently linked to the oligodeoxynucleotide at any location. The details of forming a specific oligodeoxynucleotide conjugate is fully described in Apn1lirmntal v- nmle YTIF WO 91/19730 PCr/us91/03886 -16-

EXAMPLES

The following Examples illustrate this invention (its compositions, processes and utility) with relation to specific metals, imidazole groups, ligands, nucleosides, nucleotides and oligodeox-'nucleotides.

Specifics set forth in these Examples are not to be taken as limitations on the scope of the invention or to the applicable agents, elements or features of the invention.

EXAMLE I This Example shcws how metal complexes and other compounds are screened for RNA hydrolysis activity.

Stock solution (1mM) of various transition metal complexes (shown in Table I) were prepared in 20mM HEPES buffer pH 7.1. The assay mixture in a final volume of contained 1,33AM metal complex, 63AM poly(A),.,a and HEPES buffer. At time 0 hours a i000L of the mixture was removed and analyzed by Applicants' Assay.

The reaction mixture was then incubated at 37"C. for 18 hours after which time a second 200AL was removed and assayed. Summarized in Table I are the RNA hydrolysis active and inactive transition metal complexes and corresponding cleavage obtained as determined by Applicants' Assay.

WO 91/19730 WO 9119730PCfI/US9I /03880 -17-

TABLEJ

Active t Cleavacre -Inactive %Cleavacre Cu (trpy) 2 75 CU (bpy) 2 2 0 CU (bpy) 43 Ni- (CR) 2 0 Zn-N-Me-(CR) 2 20 [CuCR(CH 2 3 puCR] 4 0 Cu-2,2-CR 2 22 Cu(EDTA) 0 Zn(EDTA) 0 Zn(NTA)1-' 0 Abbreviations: trpy 2,2':6',2"1-terpyridine; bpy- 2,2' -bipyridine; N-Me-(CR) (N-methyl) 12-dimethyl- 3,7,11, 17-tetraazabicyclo[11 heptadeca-1 (17) ,2,11,13,15-pentaene; CR 2,12-dimethyl-3,7,ll,17tetraazabicyclo (1l.3.l]heptadaca-l (17) 11,13,15pentaene; 2,2-(CR) 2,10-dimethyl-3,6,9,12tetraazabicyclo(9.3.1]pentadeca-1 (15) ,2,10,12,15pentaene; EDTA ethylenediaminetetraacetic acid; NTA nitrilotriacutic acid; CR(CH 2 3 (CR) 7,7' (1,3propanediyl) -bis[2, 12-dimethyl-3-7, 11, 17tetraazabicyclo(11.3.l]heptadeca-1 (17) ,2,1l,13,15pentaene.

Summarized in TABLE 2 are compounds known to Applicants which are found not to be effective in RNA hydrolysis according to Applicants' Assay.

JALE 2 Compoundj %cleavagre The identity and structure of the compounds in Table 2 are shown Figure a.

WO 91/19730PC/SI038 PCT/US91/03886 -18- This Example shows that under conditions where a Cu(bpy) 2+complex substantially hydrolyzes RNA, it does not degrade DNA.

The observed cleavage of RNA by various Cu(bpy) 2 complexes was hydrolytic and not oxidative. This was demonstrated by comparing the reactivity of the Cu(bpy) 2 complexes with both DNA and RNA. A stock solution of DNA [poly(dA) 2 15 J was prepared by dissolving 25 uniits of the DNA in l.OmL of 20M HEP2S buffer pH 7.1. The reaction mixture contained in a total volume of 63gM of the DNA, 157MM bipyridine, 157AM Cudl 2 and HEPES buffer. The solutions were incubated at 37*C for 48 hours after which time they were assayed by ion exchange HPLC. Identical conditions were used in the reaction of the Cu(bpy) 2' complex with RNA [poly(A) 12 1 8 Figure 6 contains the HPLC analysis of the reactions of the Cu(bpy) 2 complexes with the DNA and RNA. After 48 hours the RNA is extensively hydrolyzed.

By contrast, the DNA substrate showed no evidence of degradation. It has been reported that both RNA and DNA are oxidatively cleaved by 1,10-phenat1hroline-copper(II) at similar rates B. Chen at al, J. Am. Chem. Soc.

1988, 110, 6570-6572). Consequently, one would expect to see extensive cleavage of the DNA by the Cu(bpy) 2 complex if an oxidative mechanism was operative.

EUR.LIL

This Example shows the attachment of bipyridyl ligand (bpy) to the 3' position of 21-deoxy-thymidine nucleotide as outline in Scheme 1.

'-O-DMT-2 '-deoxy-thymidine-3'-O- -cyannethyl N,N-diisopropyl phosphoramidite (0.4 gin., 0.537 mmol) was dissolved in anhydrous CH 3 CN (5mL) -under N 2 and tetrazole (0.112 gin., 1.61 uinol) was added. DMT is 4,4'-dimethoxytrityl. The resulting mixture was stirred at room temperature for 15 nifiutes. An acetonitrile solution (5mL) of 4 '-methyl-4- (hydroxybutyl) -2,2 bipyridine (0.130 gin., 0.540 mmol) was added and WO 91/19730 WO 9119730PCrIUS9I /03880 -19after l-hr. the mixture was concentrated in vacuo to yield a glass of compound 5'-0-DMT-2'-deoxy-thyMidine- 3 '-methyl(2,2 '-bipyridin)-4-yl~butoxy)- cyanoethoxyphosphine Compound was dissolved in

CH

2 C1 2 (3mL) cooled to 0OC in an ice bath and t-BuOOH in 2,2,4,4-tetramethylpentane (0.643 mL, 1.93 nimol) was added. After 20 min. the mixture was concentrated in vacuo to yield a glass of 5'-O-DMT-3'-[4-[4'-ethyl(2,2'bipyridin)-4-yljbutyl- -cyanoethy1 phosphateJ-2 '-deoxythymidine Compound (0.382 gm., 0.432 mmol, 80.4%) was eluted from an Alumina column (neutral) using MeOH in CH 2 C1 2 Compound (0.382 gin., 0.432 mmzol) was dissolved in aqueous NH 3 (l0mL) and left to stir at room temperature for 6 hours. The mixture was concentrated in vacuo u~sing EtOH to remove water. The residue was treated with 25% CF 3 COOH in CH 2 C1 2 (5inL) for 15 minutes.

After removing the volatile components in vacuo the residue was dissolved in water (IOinL) and the aqueous layer washed with ether (2x5mL) and CH 2 CL2 (2x5inL). The aqueous layer was concentrated in vacuo to yield the desired deprotected nucleoside 3 '-methyL(2,2 bipyridin) -4-yl~butyl-phosphatej -2 '-deoxy--thyinidine amnmonium salt (0.192 gm., 0.354 inmol, 82%).

LEM

This Example shows the titration of compound w~ith Cud 2 to form 31-(4-[41-methyl(2,2'-bipyridin)-4yl ]butyl-phosphate) -2 '-deoxy-thymidine amnmonium salt copper(IX) (Figure 2).

A 1mL aliquot of a 53.4MM solution of compound in 20mM HEPES buffer having a pH of 7.1 is placed in a quartz cuvettn and aliquots of a 1.178mM stock solution of CuC1 2 in water was added. Changes in the visible spectrum were monitored between the wavelength of 240 and 380nn. The addition of cuC1 2 causes the band at 276nm to decrcase with con6oinitant increase in absorbances at 302 and 312nin. The changes occur with an WO 91/19730 WO 9119730PCT/US9I /03886 isosbestic pint at 289nm and are characteristic of coordination of Cu 2 to bipyridine.

A similar titration with the solution of 3'thymidine monophosphate without the bipyridine ligand showed no changes in the visible spectrum over the noted region.

E2U=LE- This Example shows the hydrolysis of RNA [poly(A) 12 by a nucleotide covalently linkced throug1 the 3' position to a metal complex (Figure 3).

For the reactions of metal complex-nucleotide (nu~cleoside) conjugates, Applicants' HPLC Aosay described above was modified. These compounds are not as reactive as the free metal complexes, thus, the reaction time was extended to 48 hours.

In a total of 500ML, the reaction mixture contained 157AM of compound 631iM RNA, 157gM Cu(S0 4 and 20 mM HEPES buffer pH- 7.1. Using this mixture, compound was formed under the conditions set forth in Example IV. At time zero, a lOOML aliquot of the reaction 'mixture was Irtmoved and immediately analyzed by Applicrants' HPLC Assay'. The reaction mixture was incubated at 370C for 48 hours after which time a second aliquot was removed and assayed. It was found that ,K 9 RNA substrate was clearly hydrolyzed by compound (6) (Figure 3).

A~ control reaction carried out in the same manner except in the absence of the Cu(S0 4 showed no hydrolysis of the RNA even after the 48 hour incWbation.

EXAMLEVI

This Example shows the attachment of bipyridyl ligand (bpy) to the 5' position of 2'-deoxy-thymid~ine nucleotide as outlined in Scheme 2.

Preparation of 5'-(4-[4'-methyl(2,2'-bipyridin)- 4-yl) butylj -methylphosphate-3' -0-acetyl-2 '-deoxythymidine C10): a mixture of A '-4-methy112.2Vbipyridin) -4-yl Jbutyl-methyl-N ,N-diisopropyl phosphoramidite 0.101 gin., 0.25 minol) and tetrazole WO 91/19730 W0919730P'/US9I /03880 -21in lmL of THF was stirred at room temperature for minutes. 3 '-0-acetyl-2'-deoxythymidine 071 gin., 0.25 mmol) dissolved in CHCl 2 (lmL) was added to the reaction mixture and tlhe solution was left stirring for 60 minutes. The mixture was then filtered to remove tetrazole which precipitated out. The solid was washed with acetonitrile and CH 2 C1 2 (5mL) and concentrated to yield 51--(4-[41-uiethyl(2,21-bipyridin)4-yl) butoxy~methoxyl -3 '-0-acetyl-phosphine-2' -deoxy-thymidine as a glass. The compound glass was dtissolved in MeOH (ImL), cooled to 0'C and a 3M solution of t-butyl hydroperoxide in 2,2,4,4-tetramethylpentane (0.3 mL, 0.9 mmol) was added to the stirred reaction mixture. After min. the ice bath was removed and the mixture stirred at room temperature for 20 minutes. The mixture was concentrated to a glass and flash taromatographed on an alumina column (TLC grade). The desired compound (0.071 gmn., 0.118 inmol, 47%) eluted off the column using a gradient of CH 2 Cl 2 to 10% MeOH in CH 2 C1 2 as an eluant.

Preparation of 5'-[4-[41-'methyl(2,21-bipy'ridin)- 4-yl )butylphosphateJ -2 '-deoxy-thymidine triethylammionlun salt to compound (10) (0.071 gmn., 0.118 mmiol) dissolved in CH 2 C1 2 (UiL), 25% 14aoMe in MeOR (0.05 mL, 0.12 inmol) was added. The mixture was stirred for min. at room temperature. After 15 min. the reaction was quenched with glacial acetic acid (0.06 gmn., 0.12 mmiol). Dichioromethane (5OmL) was added anid the organic layer washed with saturated NaHC0 3 solution (2x2OmL) and water (10mL). The organic layer was dried over anhydrous N%,S04 and concentrated in vacuo to a glass (0.064 gm., 0.114 inmol, The glass was dissolved in 0.5 mL thiophenol~dioxane:triethyanine a commercial deprotection reagent by Sigma Chemicals, and left to stir for 90 minutes. The mixture was concentrated in vacuo to a glass and the residue dissolved in water (10mL). The aqueous, layer was washed with petroleum ether (2xlOmL) to remove traces of thiophenol. F±ial purification was carried out by RP C- WO 91/19730 WO 9119730PCr/US91/03886 -22- 18 Sep-Pak column and eluting the desired compound (11.) (0.069 gm., 0.106 mmol, 90%) with H 2 0:CH 3 CN This Example shows the titra~tion of compound (2.1) prepared in Example VI with CuC1 2 to form methyl (2,2 '-bipyridin) -4-ylJ -butyl-phosphate) -2 '-decxcythymidina triethylammonium salt copper(II) (12).

The procedure described in Example 1I was followed. Changes in visible spectrux, similar to those shown in Figure 3, characteristic of coordination of copper(II) to bipyridine were observed. Titration of showed no changes in the visible spectrum over the range 240-380nm.

ZXL9 VI This Example shows the hydrolysis of RNA [poly(A) 218 by a nucleotide covalently linke4. through the 5' poaition to a transition ueta complex.

An identical procedure was used as described in Example IV except that compound (12) was the hydrolysis agent (Figure 4).

This Example shows the attachment of bipyridyl ligand (bpy) to the 5- position of the uracil in a uridine nucleoside as outlined in Scheme 3.

Preparation of [4-[4'-mathyl -bipyridirj -4-ylJ -l-oxobutyJliamino] ethyl 3amino] -3oxopropyl]-21-deoxy-uridine a solution 'of 5-C3- -aminoethyl) amino] -3 -oxopropyl 3-5 '-O-DMT-4eoxyuridine (0.322 gmn, 0.5 minol) in CH 3 CN (5x.L) and Et 3

N

(0.2mL) was cooled to 0OC in an ice bath an6 4-E3-carbo- (p-nitrophenoxy) propyl] -4 '-methyl-2, 2'-bipyridine (14) (0.566 gmn, 1.5 inmol) was added to the stirred reaction mixture. After 15 min. the ice bath was removed and the mixture was stirred at room temperature for 24 hours.

The reaction mixture was diluted with 2OimL CH 2 Cl 2 and water (10mL) The aqueous lak'er'was washed with CH 2 C1 2 (2x2OmL) The Alried (MgS04) organic layer was concentrated in vacuo and flash chromatographed on an WO 91/19730 WO 9119730PCT/US9I /03880 -23alumina (neutral) column eluting with a gradient of

CH

2

C.

2 to 6% EtOH in CH.C1 2 to remove p-nitro-phenol. The desired product eluted off the column using MtON along with C-3 bpy acid. Compound (15) (0.211 gin., 0.24 mmol, S1 45%) was purified by RP HPLC using a linear ternary gradient flowing at 6 mL/minute. Solvent A (0.1M) EEt 3 NH)OAc was kept constant while M9CN and H 2 0 were varied.

Prepa,tioii of bipyridin3-4A-yl]-l-oxobutyl]amino)ethyl]auino]-3oxopropyl]-21-deoxy-uridine a solution of the nucleoside (15) 1.0.150 gmi., 0.17 mnmol) in CH 2 C1 2 was treated with 10t CF 3 COOH in CH.Cl. (5mL) for minutes. The mixture was concentrated to a glass and dissolved in water (IOmL). The aqueous layer was washed with CH 2 C1 2 (2xlOmL) and compound (0.093 gm., 0.16 minol, 94%) was purified by RP HPLC using a linear ternary gradient flowing at 6 mL/minute. Solvent A (0.1M) [Et 3 NXJOAc was-kept constant while MBCN and Hi 0 were varied.

This Example showo the titration of compound (16) with CuCd 2 to form 5-[3-E[2-[(4-(41-methyl[2,2'bipyridin) -1-oxobutyl 3amino] ethyl]aninoj -3oxopropyll -deoxy-uridine copper (II) (17).

The procedure described in Example III was follo-vad. Changes in visible spectrum similar to those shown in Figure 2 and characteristic of coordination of copper(II) to bipyridine were observed. Titration of uridine shovied no changes the visible spectrum over the range 240~-380=i.

EX&MLE X1 This Ex'ample shows the hydrolysis of RNA lpoly(A) 12 18 J a metal complex covalently linked 3S through the 5- position of uracil in a uridine nuc~leoside.

An identical procedure was used as described in Exaiple IV except that compound (17) was the hydrolysis WO 91/19730 WO 9119730PCTIUS91 /0388' -24agent. Hydrolysis of the RNA is evident as shown in Figure This Example shows the preparation of various terpyridine (trpy) derivatives (Scheme 4) which can be attached to nucleotides as described in Examples IV and V and which have been previously shown in Example I to be active RNA hydrolysis catalysts.

Preparation of 4'-(3-formylpropyl)-2,2':6' ,2"1terpyridine a 100 mL three necked RB flask, equipped with a rubber septum, a teflon coated stir bar and a gas inlet adaptor was f lushed with 4 1-methyl- 2t2':60,2"1-terpyridine (18) (0.494 gmn., 2 inmol), prepared by literature methods (see K. T. Potts, et al, J. Am. Chen. Soc., 1987, 109, 3961-3967) was dissolved in dry THF (lOmL) and was syringed into the flask. The reaction mixture was cooled to -78*C and LDA (1.6 mL, 2.4 mmol) was added via syringe. The resulting dat.s; brown mixture was stirred for 2 hr. at -780C. 2-(2bromoethyl)-1,3-dioxolane (0.434 inL, 2.4 mmol) was syringed into the reaction mixture, stirred for 1 hr. at -78'C and allowed to warm to room temperature overnight.

The mixture was poured over 10 ml brine and the aqueous layer was extracted w ith CH5,C1 2 The extracts were dried over MgSO4 and evaporated to dryness to yield *he crude acetal. The acetal was hydrolyzed with 1M HM1 (l~mL) by heating to 50-600C for 2 hours. The solution was then neutralized with aqueous NaHCO 3 and extracted with

CH

2 C1 2 The extroots were dried over MgSO 4 and evaported to dryness to yield the crude aldehyde., RP'rification by flash chromatography (neutral alunilia,

CH

2 C1 2 elution) gave pure aldehyde (19) (n1.339 gin., 1.12 mmol, Preparation of (4-hydroxybutyl) 6' ,2 terpynidine the aldehyde (19) (0.303 gmn., I. xmm~ol) was dissolved in absolute ethimol (5xnL) and sodium borohydride (0.05 gin., I. iniol) was added'at room temperature. After stirring for 30 min. the mixture was WO 91/19730 WO 91/19730 T/US9I /03880 poured into 10mL brine~ and extracted with CH 2 Cl.

(3XIOML) The extracts were dried over MgS0 4 and evaporated to dryness to yield the desired alcohol (0.264 gmn., 0.86 minol, 86%).

Compound (20~ is analogous to compound in Scheme 1 and can be attached to the 3' position of thymidine in a siiiar fashion as described in Example 11.

Preparation of 4 '-(4-bromobutyl) 2"-terpyridine the alcohiol (20) (0.200 gm., 0.65 minol) was diasolved in 5mL H~r and reflnxed for 6 hours.

After cooling to rooL temperature the inixt\vr* was pour:ad over 20 gr,. of crushed ice, basified with saturated aqueous solution 2 f Na 2

CO

3 and extracted with CHzClz. The extracts were dried over (Mg'SO 4 and evaporated to dryness to yield the bromo derivative (21) (0.186 qm., 0.51 inmo3, 78%).

Preparation of (4-phthalimidobutyl) 2,21:61,2"1-terpyridine the bromo co~mpound (21) (0.186 gmn., 0.51 mmiol) in LVMP (2mL) was added to a suspension of potassium phthalimide (0.095 gm., 0.51 inmol) in DHF (lmL). The mixture was stiv~d for 2 hr.

at 60-600C. After cooling the reaction mixture was poured into water l0inL) and the resulting mixture was thoroughly extracted with CHCl 3 (3x25mL). The organic layers were combined, washed with 2OmL of 0.2 M NaOH, water (2OmL) and dri~ad over Removal of solvent under reduced pressure gave a thiick oil.

Recrystallization from etnanol gave compound (22) as a white crystalline solid (0.208 gin., 0.48 inmol, 95%) mp 1260C.

Pronaration of (4-aminobutyl) 211terpyridine phthalimide derivative (22) (0.208 gin., 0.48 was suspended in 7xnL EtOR and treated with hydrazine hydrate (88 mg., 0.48 uimol). The mixture was ref luxed for 6 hr., cooled~tb room temperature, poured into brine (lOinL) and basified with 50% w/w NaOH to pH 2. The mixture was thoroughly extracted with WO 91/19730 WO 9119730PCI'/US91 /03880 -26-

CH

2 C1 2 (3XlOVL). The rgaric layers were dried over Na 2

SOI

4 Removal of solvent undar reduced pressure gave the desired product (23) (0.130 gm., 0.43 umol, 89%).

Compound (23) can be attached to the 5' position of 2'-deoxy-thymidine by literatu.a procedur-es (see B.

C. F. Chu et al, DMA 1985, 4, 327-331).

EXAMLE XI This Example shows a variety of nucleosides and nucleotides which have groups A~ppended on the 3' and position of 2'-deoxy-thymidine and 5- position uracil in 2'-deoxy-uridine and which are not active at hydrolyzing RNA [poly(A) 2 1 under the conditions of the HPLC assay (Table 2).

No hydrolysis of the RWA was observed with the compounds shown in Table 2 when assayed in the absence of CU(S0.) under the conditions described in Example IV.

This 'Example shows the attachment of bipyridine ligand to a 14mer oligodeoxynucleotide (30) as shown in Scheme Preparation of 4-(3-carbo-N-hydroxysuccin- imidopropyl) -4 '-Aiethyl-2,2 1-bipyridine 4-(3-carboxypropyl)-4'-methyl-2,2'-bipyridine (28) (1.0 gmn., 3.9 minol) and N-hydroxysuccinimide (0.494 qgm., 4.3 mmol) were dissolved in EtOAc (IOML) and the mixture was cooled to 0OC in an ice bath. Compound (28) was prepared in accordance with the teachings if L. Ciana et al, J. Org. Chum. 1989, 54, 1731-1735. Dicyi 2Iohexylcarbodiimida (DCC) (0.804 gin., 3.9 mmol) was added in small portions and the mixture was stirred at 0QC for minutes. The ice bath was removed and the mixture was allowed to stir at room temperature for 12 hours.

The resulting precipitate war) filtered of f and the filtrate concentrated in vacuo. The residue was dissolved in CH2C1 2 (lO0mL) washed with water (50mL) and dried over Mg (SO 4 The dried organic extract was concentrated in vacuo to yield compound (29) (0.827 gm., 2.3 mmol, 59%).

WO 91/19730 PCT/US91/03880 -27- Preparation of oligodeoxynucleotide-bipyridine conjugate to an aqueous solution (20mL) of the oligodeoxynucleotide (30) was added a solution of compound (29) in CH 3 CN (20mL). The pH of the mixture was raised to 9.3 by the addition of Et 3 N and the mixture was stirred overnight. The oligodeoxynucleotide-bipyridine conjugate (31) was purified by anion exchange HPLC.

EXAMPLE XV This Example shows a sequence-directed cleavage of tRNATyr by an oligodeoxynucleotide-bipyridine copper(II) (32) complex in accordance with this invention as outlined in Scheme 6.

A 101M stock solution of oligodeoxynucleotidebipyridine conjugate (31) was prepared by dissolving 6.1 units of compound (31) in 500 L of 20 mM HEPES buffer having a pH of 7.1. A 25.9pM stock solution of the tRNAYr substrate was prepared by dissolving 10 Units of tRNATYr in 500AL of 20 mM HEPES buffer having a pH of 7.1.

The cleavage reaction contained in a total of 600AL, 1.29MM tRNA'yr, 12.9AM Cu(trpy) 227MM Cu(SO 4 6.4AM compound 50mM NaCl and 50mM HEPES buffer having a pH of 7.8. Initially the tRNA'~, compound NaCI and buffer were combined and heated to 65*C for 4 min.

in a water bath. The reaction was removed and immediately placed on dry ice. The mixture was allowed to thaw at 0*C after which time the Cu(S04) and the Cu(trpy) 2 complex were added. Applicants have shown in Example III that the copper(II) coordinates to the bipyridine ligand exclusively forming in this case the oligodeoxynucleotide-metal complex conjugate The reaction was heated at 37*C and 100L aliquots were removed at times 0, 17 and 28 hours. Analysis of the aliquots by polyacrylamide gel electrophoresis revealed three distinct cleavage sites adjacent to the targeted sequence as shown in Figure 6. These bands appeared in a time-dependent fashion and control reactions were devoid or showed significantly reduced cleavage in these regions.

WO 91/19730PC/SI03& Pcr/usgi/0388d -28- The predicted position of RNA cleavage based on the hybridization of compound (32) to tRNATPr would produce a fragment 51 nucleotides in length (Scb'ame This fragment was observed. Two other fragments also appear from the reaction. These are attributed to cleavage at sites brought close to the reactive group in compound (32) due to three dimensional folding of the tRNA Tyr molecule. Thus, sequence-directed cleavage of tRNA~yr was demonstrated.

I

This example shows the synthesis of the diimidazole containing nucleoside aminoJ-l-oxo-3-f[.H-imidazol-4-yl)propyl~aminoJ-l-oxo-3- [lH-imidazol-4-yl]propyl] amino]ethyl]amino3-3oxopropyJ.J-2'-deoxy-uridine, as outlined in Scheme 7.

The synthesis of compound (11) has been previously described (Dervan et al., Proc. INatnl. Acaed. Sdl. USA 1985, 82, 968.).

The nucleoside 5' -0-DMT-5- (3-f C(2-aminoethyl) aminoJ-3-oxopropylj-2'-deoxy-uridine (1A) (1.288 g, mmol) was dissolved in dry dichloromethane (10 niL) and was cooled to 0OC in an ice bath. Fmoc-L-His(Tr)-O-pfp (3.16 g, 3.0 iumol) was added to the stirred reaction mixture. Triethylamine (0.28 ml, 2.0 mol) was added to the solution and the mixture was stirred at room temperature for 8 hours. The reaction mixture was concentrated and purified by flash chromatography an a silica gel column eluting with a gradient of 100% CH 2 Cl 2 to 12% EtOR in CH 2 Cl 2 The nucleoside 5'-O-(Bis(4methoxyphenyl)phenylmethyl]-5-[3-((2-f (2-f ((91-f luoren- 9-yl-methoxy) carbonyl] amino] (triphenyl methyl) 1H-imidazol-4-yl]propyl] aminojethyl) amino] -3oxopropyl]-21-deoxyuridine (2A) (1.94 g,l.56-mmol, '78%) eluted off the column using 10% EtOB in CH 2 C1 2 The nucleoside P2A) (1.94 g, 1.56 mmol) disaolved in CH 2 C1 2 (10 MiL) was treated frr 3 hours at room temperature with Et2KH (10 niL) and the mixture was concentrated to a glass. The residue was flash WO 91/19730 Wo 9119730PC1'/US9I /03880 chromatographed on a silica gel column. The deprotected impurities were eluted of f the column using CH 2 C1 2 and the nucleoside (Bis (4-methoxypheny.) phenylme4,hyl] 5-[3-f 2-famino]-3-fl-(triphenylmethyl)-1H-imidazol-4yl~propyl~amino~ethyljamino]-3-oxopropyl]-2 '-deoxyuridIine (3A) (1.43 g, 1.4 mmol, 90%) was eluted off with EtOH in CH 2 Cl 2 A solution of (3A) (ig, 2. 0 mmol) in dry dichioromethans (lOmL) was cooled to 0OC in an ice bath and Fmac-L-His(Tr)-.-pfp (3.16 g, 3.0 mmol) was added to the stirred rsaaction mixture. Triethylamine (0.28 ml, 2.0 mol) was added to the solution and the mixture was stirred at room temperat,,ure for 8 hours.

The reaction mixture was concentrated and flash chromatographed on a silica gel column eluting with a gradient of 100% CH 2 C1 2 to 15% EtOH in CH 2 C1 2 The nucleoside (bis (4-methoxyphenyl) phenylmethyl] 13-f (2-f 2-f (2-f f(9H-fluoren-9-yl-methoxy)carbonyl]amino] -l-oxo-3- 1- (triphenylmethyl) -1H-imidazol-4yl]propyl] amino]-1-oxo-3-'fl-(triphenylmethyl) -11imidazol-4-yl~propyl ]amino] ethyl] amino] -3-oxopropyl] 2'-deoxyruridine (4A) (1.94 g,1.56 mmol, 72%) was eluted off with 12% EtOH in CH 2 Cl 2 mp 156-80C.

The nucleoside (41) (0.4 g, 0.246mmo1) was dissolved in CH 2 Cl. (5 ml) and was treated with dieathylamine (5mL) and left to stir for 9 hours. The mixture was concentrated and the residue flash chromatographed on a silica gel column. Thz compound, eluted of f the column using 10% NH 3 in EtOH was stirred with a solution of 15% CF 3 COOH in CH 2 Cl 2 (5 ml) After minutes, the mixture was concentrated. The residue was suspended in CH 2 Cl 2 (25 ml) and water (10 xml) The aqueous layer was washed with CH 2 C1 2 (2x5 ml) and ether ml) and con~centrated to give the diimidazole nucleoside conjugate 5-f 3-f(2-f (2-f (2-amino)-l-i ,xo-3flH-imidazol-4-yl]propyl]amino -1-oxo-3-f1H-imidazol-4yl]propyl]amino]ethyl]amino]-*noxopropyl]-2 .deoxyuridine (5)-(0.120 g, 0.194 mmols, 79%).

WO 91/19730 PCUS91/0388Y EXAMPLE XVII This example shows the cleavage of a 172mer RNA fragment by compound (5A) and that the mono-imidazole compound 5-[3-[[2-[[2-amino-3-(lH-imidazol-4-yl)-loxopropyl]amino]ethyl]amino]-3-oxopropyl]-2'-deoxyuridine (6A) (Bashkin, Gard, and Modak, J. Org. Chem< 1990, 55, 5125) does not hydrolyze the RNA fragment under identical reaction conditions (Figure 9).

Extensive precautions were taken to avoid RNase contamination in the hydrolysis reactions. All buffers were made with distilled-deionized water which was treated with diethylpyrocarbonate vol/vol) and hydrolysis reactions were run in sterilized polypropylene tubes. Analysis of the cleavage reactions were performed with denaturing polyacrylamide gel electrophoresis using standard techniques (D'Alessio, J. M. In Gel Electrophoresis of Nucleic Acids A Practical Approach; D. Rickwood and B. D. Hanes Eds. IRL Press Limited: London 1982; p. 173-196.) Uniformly 32

P

radiolabeled RNA substrate was generated by runoff transcription with bacteriophage SP6 DNA-dependent RNA polymerase using standard techniques (Maniatis, T.; Fritsch, Sambrook, J. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: New York, 1982). HEPES buffer, purchased from Sigma Chemical Co, (St. Louis), was used without further purification. All electrophoresis reagents used were RNase free.

A stock solution of 3 P labeled RNA was prepared by dissolving approximately 40gg of RNA in 90 IL of HEPES buffer pH-7.1. Stock solutions of 4.9 mM, and 3.1 mM were prepared in 20 mM HEPES buffer.

The control reaction contained in a total volume of ML, 4.5 Mg 3P labeled RNA which was diluted to volume with HEPES buffer. The reaction with agent contained in a total volume of.90 AL, approximately 23 Ag of 32 P labeled RNA and 1 mM compound The reaction with agent (6A) contained in a total WO 91/19730 PCT/US91/03880 -31volume of 50 ML, approximately 9 sg of 32 P labeled RNA and 2 mM compound All reactions were run at 50' C for a total of 2 hours after which time 20 pL was removed and immediately frozen on dry ice. In the case of reaction a single point was taken at 2 hours. In the case of reaction 20 AL aloquats were removed at time 0, 0.5, 1, 1.5, and 2 hours. In the case of reaction 20 AL aloquats were remove at t 0 and 2 hours. The samples were loaded onto PAGE gels 7 M urea) and were run for 4 hours at 900 V. Gels were developed using standard autoradiography techniques (Maniatis, Fritsch, Sambrook, J. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: New York, 1982.).

The results shown in Figure 9 clearly demonstrate that reactions #2 shows nearly complete cleavage of RNA (Lanes 4, 5, while reactions #1 (Lane and #3 (Lane #7 and 8) are devoid of RNA cleavage. The fact that reaction #3 shows no cleavage even with 2 mM total imidazole concentration indicates that incorporating two imidazoles into the same molecule, such as in compound provides for efficient cleavage of RNA.

EXAMPLE XVIII This example shows the incorporation of imidazole-nucleoside conjugates into di-, tri-, and oligodeoxynucleotides. These techniques may be applied for the incorporation of suitably protected conjugates into oligodeoxynucleotides.

Two complementary approaches were explored for the synthesis of imidazole-DNA conjugates: solutionphase phosphotriester chemistry and solid-phase phosphoramidite chemistry. Both approaches employ the previously reported (Bashkin, J. V ct al., J. Org. Chem.

1990, 55, 5125) compound whicZh is a protected form of compound Thus, Schete 8 shows the use of (7A) in the preparation of dinucleotidi Xp,. (compound using the o-chlorophenylphosphatt ester technique WO 91/19730 PCT/US91/03880 -32- (Reese, C.B. Tetrahedron 1978, 34, 3143). Intermediate phosphodiester (SA) was prepared in 82% yield, and characterized by FABMS. The fully-protected phosphotriester (9A) was prepared in 49% isolated yield; FABMS showed ions corresponding to M+3Li, M+2Li, M+Li, and M+3Li-Boc. After deprotection of (9A) and purification of the product by reverse phase HPLC, the desired dinucleotide (10A) was obtained.

Scheme 9 shows the preparation of phosphoramidite (11A) and its use in the solid-phase synthesis of the dinucleotide XpT, compound (12A). Phosphoramidite (11A) has the characteristic 31 P resonances at 149.4 ppm assignable to its two diastereosars; it was further characterized by FABMS and high resolution mass spectra.

The IH NMR peak assignments of (15A) are given in the experimental section. To test its general applicability, phosphoramidite 11 was then employed in the synthesis of (13A), an 11-mer oligodeoxynucleotide with the modified nucleoside at an internal position.

The sequence prepared was 5'-TATCTTCTXAC-3', where X indicates the imidazole-containing nucleo5?-e analogue.

Melting points were taken in Kimax soft glass capillary tubes on a Melt-Temp melting point apparatus equipped with a calibrated thermometer. All nuclear magnetic resonance spectra were recorded on Varian spectrometers at 25'C. The 1 H and 13 C spectra were measured on a VXR-400 while the 31 P spectra were obtained on a XLA-200. The proton spectra resulted from Fourier transformation of the accumulated scans, consisting of 30016 data points in a 8 KHz spectral width rith an acquisition time of 1.876 sec. Data were acquired with a 35' pulse (10 ms) and, where necessary, the strong H 2 0 resonance was presaturated for 3.0 s. The free induction decays were zero filled to 32K and 0.5 Hz line broadening applied to the data prior to Fourier transformation. The significant'chemical shifto are reported in ppm (d units) downfield from TMS and the Jpc values are given in Hz. All compounds are more than 98% WO 91/19730 WO 9119730PCT/'US9I /03880 -33pure by 1 3 C and 1H NMR spectroscopy. Exchangeable protons are labeled (ex).

The high resolution mass spectra (HPMS) were razorded on a Finnegan/MAT9O spectrometer while the )7'AB+ low resolution spectra were run on a VG 40-250T spectrometer. The FAB matxix was a saturated solution of Lil in 3-'nitrobenzyl alcohol, which is especially useful for acid-labile, protected nualeosides. Thin layer chromatography was performed on Baker-Flex Silica' gel 1B2-F plates and spots visualized by irradiation with UV light (254 nm).

Preparative TLC was carried out by centrifugal TLC on a Chromatotron (Harrison Research) using silica gel plates (Analtech).. Column chromatography was performed on Silica gel (Merck SG-6Q, 230-240 mesh). P HPLC was carried out on a Ailtech Econosil C18 preparative column (10mn, 250 x 22.5 mm) for di- and tri--nucleotides using a linear ternary gradient flowing at 6 mL/ min: Solvent A (0.1M[Et 3 NHJOAc) was kept constant at 25%, while B (Mt6Ck) anid C (H 2 0) were varied as follows, where time is in min: (time,%B, (0,5,70) (33,35,40) (45,70,5). Longer oligodeoxynucleotides were purified on a Nucleogen-DEAE 60-7 preparative column using a linear binary gradient flowing at 1.5 mL /min: Solvent A (80% 0.1M NaOAc, 20% MeCN) and Solvent B (20 MeCN, 80% IM LiCl and 0.02M NaOAc) were varied as follows, where time is in min: (tlme,%A, (0,100,0) (33,0,100) (43,100,0). The HPLC was monitored simultaneously at 260. and 400 nm.

The compounds p-nitrophenol, L-histidine (sigma ChemicalL,, Fmoc-L-Lys (Boc) -Opfp (Pharmacia), dicylohexyl carbodiimide, diethylamine, diisopropylethylamine (Aldrich), Chloro-N,N-.

diisopropylainino- -cyanoethoxy-phosphine (AEN) were used without any further purification.

P~r22aration-of 5'-0-rBis(4-mgthgyp=heny~jzphenylmethv11-5-r3-r2-r r2-r r oren-.yl..

rethoXylcar onyllaminol-3-r T1-(2.2-dietyJ- WO 91/19730 WO 9119730PCF/US9I IO3886' -34ethoxvj carbonyl 1 -3H-imi~dAzol-4-vl 1 am~ino I ethyl I amino-3-oxo]2rg]2l 1 -3 1 -ro-r~chro~hanylphodih~iester-2-deoy-ridine (WI).

o-Chlorophenyl phosphorodichioridate (0.897 g, 3.66 mmol) was weighed into a two-necked pear-shaped flask and dissolved ir, acetonitrile (10 mL). 1,2,4- Triazole (0.556 g, 8.052 mmol) and triethylamine (1.02 mL, 7.32 umol) were added to the reaction vessel, and the mixture stirred at room temperature for 20 minutes.

The nucleoside (7A) was dissolved in acetonitrile and 1-Me-imidazole (0,1 mL, 4.88 mmol) was added to the stirred solution. This reaction mixture was added to the phosphorylating mixture in the pear-shaped flask and stirred at room temperature for 210 minutes. The reaction was monitored on TLC, and after all of the starting material was consumed, the mixture was quenched with triethylamine (3.06 mLf 21:06 mmol) and water mL) to give a homogeneous solution. The solution was stirred for 10 minutos akid then concentrataid. The z~sidue was dissolved in dichloromethane (25mL) and washed with sat. NaHCO 3 (25 mL). The aqueous layer was washed with dichloromethanoi (2x2OinL), and the combined organic extracts were driod over MgS04 and concentrated to a glass. The glassy material was dissolved in dichloromethane (l0mL) and precipitated from pet. ether (500mL). The solid phosphodiester (GA) (1.16 g, 0.91 mmcl, 82%) was collected by centrifugation and dried in a vacuum desiocator. FABMS n/z 1171, (M 1071t (M H Boc).

Preparation of Dimer X=DC (9k).

The phosphodiester 8 (0.427 g, 3.36 mmol) was.

dissolved in dry pyridine (5 mL) and 4-N-3'-O-diacetyl- 21-deoxycytidine (0.095 g, 3.05 mm01) was added to it and the pyridine removed under reduced pressure. The process of addition and removal of pyridine was carried out twice to remove traces ofmolsture. l-(2mesitylenesulfonyl) -3-nitro-l,2 ,4-triazole (0-361 g, 12.2 mmol) was then added to the solution of the two WO 91/19730 WO 9119730PCTr/US9 1/03880 nuclaosides and stirred at room temperature for 20-25 minutes. The mixture was then quenched with 1 m7A saturated solution of NaHCO 3 Dichioro-methane (lO0mL) was added to the reaction mixture after 5 minutes, and the organic phase was washed with water (50 mL). The organic extr4Lcts were dried over MgSO 4 and concentrated to yield a glass. The glass was chromatographed over silica gel and the product (9A) (0.220 go 1.5 mmol, 49%) eluted with CH 2 Cl,:EtOH 90:10. FABM- m/z 1484, (M 3Ui); 1478, (M 2Ui); 1472, (H Li); 1384, (H 3Li Boc).

Preparation of 2tnC The fully protected dimer (9A) (0.220 g, 0.15 mmol) was treated with a freshly prepared solution of N 1 N'I, 14 3

N

3 -tetramethylguanidina, (0.33 H) c 1 nd anitrobenzaldoxime in dry CII 3 CN (1.5 zL). After 3 hours at room temperature the mixture was conc,,intrated and the residue washed with ether. The solid was dissolved in aqueous NH 3 and stirred at room temperature for 24 hours.

After concentrating the solution, the resulting solid was treated for 30 minutes with 50% CFCOOH in dichloromethane (10 mL). The fully deprotected nucleoside was extracted with water (10 mL), and~ the aqueous layer was washed with diethyl et-her (2%3 mL).

The aqueous layer was evaporated to yield the deprotected nucleoside (10A) (0,110 g, 0.143 mmol, The sample was purified on a Ailtech Econosil C18 preparative R~P HPLC column. Retention time for (1.OA) (250 OD units) was 14.6 minutes on a C18 analytical column using the same inear ternary gradient, flowing at 1.5 nL/min. 31 F NMR (D 2 0) ppm 0).19 s; HR MD 2 0) d 6.25 1H, 6.3 IH, 11C); 2.45 (mn, 2H, 2'X); 2.4 (mi, 2H, 21C); 4.8 (in, 1H, 31X); 4.6 (in, 1H, 31C); 4.1(m, 1H, 41X); 4.0 (in, 1H, 41C); 3.8 (mn, 2H, SIX); 4.2 (in, 2H, 51C); 6.1 (do 1Hi, H19); 7.9 (do 1H, HIS); 7.7 IH, 116); 2.4 (2 t's, 41, I S HS); 3.2 (2t's, 4H, H9 a HIO); 4.25 (in, IH, P11); 3.4 (in, IH, H12); 7.3 (s 111, H14) 8. 4 1H, HIS) 13C NMR (D 2 0) ppm 154.2, WO 91/19730 PCY/US9I /03880 -36- 168.3, 4C0; 116.2, 5C; 141.1, 6C; 25.8, 7C; 37.4, 8C; 41.8 Q 41.2, 9C 1bC; 55.7, 11C; 30.3, 12C; 131.0, 13C; 120.6, 14C; 138.1, 15C; 178.3, 16C; 171.9, 17C; 144.9, 18C; 98.9, 19C; 159.3, 20C; 168.2, 21C; 88.3, 11X; 88.9, 1' C; 4 0. 9, 2 1X; 42. 3, 2 0C; 77. 8, 3 1X (J P 5 .1I Hz); 7 3. 3, 3 1C; 8 t7 (J CP 6. 5 Hz) 4'1X; 88. 1, 4 1C P S. 89 Hz) 64. 0, 5 1X; 67. 8, 5 1C (3 CP 5. 05 Hz) Pre~aration of 51-0-rBis(4-methoximhenyl ~hnjany etYl1-5-r3-r2-Er2-fr(2.2-dlimethylethoxv)carb2onlpmino]-3-r rl-(2 .2-di-methylethoxvl carbonvll-lH-iymidazol- 4-vil -1-oxonro2v1 1amino 1 thyl .1amino-3-oxo~rolvl1 2 -deoxv-uridine-3$-0_-(N. N-dliisopro2]v1-A]Min2-gcvanoethoXy1Dhogl~hine (11A).

Chloro-N N-dii'sopropylano-lcyanoethoxyphosphine (0.158 g, 0.81 mmol) was weighed into an H-shaped Schienc flask arnd dissolved in ac(atonitrile (.10 mL). Di-isopropylethylamine (0.196 mL, 1.52 mmol) was added to the reaction vessel and the mixture was stirred at room temperature for 20 minutes.

0Q The nuclecside (71) (0.75 g) 0.76 mmol) was dissolved in acetonitrile and added to the phosphorylating mixture and left stirring for 30 minutes. The mixture was then filtered through the fritte to the other side of the Htube, removing the amine hydrochloride. The solid was washed with acetonitrile (2x10 mL) and the combined MeCN solutipns were concentrated to yield a glass. The glass was then cliromatographe I on a s? lica gel chromatotron (2000M). The phosphoramidite (11A) (0.721 go 0.61 mmol, eluted out with CH 2 C1:EtOAc:Et 3 N 4.5:4.5:1. NMR (CD 3 CN) ppm 149.4. Two s's (diastereo isomers) FAB3MS m/z 1194,j (M 2Ui); 1188, (M Li) 1094, (H 2Li -H Eec) 994, (M 3Li H Boc) exact mass found 1188.5789, calculated for C60HWN 9 01 4 PLi 1188.5722.

PreiparAtion of XDT (12A).

The phosphoramidite (ILA) was used on the Pharmacia gene assembler to synthesize the dinucleotide -0-DHT-X"pT which ,were fully deprotected and purified on an Alltech Econosil C18 preparative RP HPLC column.

WO 91/19730 WO 9119730PCT/US91 /03880 -37- Retention time for (2A) (25 OD units) was 1.4.5 minutes on an analytical column using the same linear ternary gradient, flowing at 1.5 m.L/min. 211 N14R (D 2 0) d 6.3 (t, 111, 6.4 1H1, 11T); 2.55 (mn, 2H1, 2.4 (in, 2H1, 21T); 4.65 (mn, 1H1, 31T); 4.85 (mn, 1H, 4.2 (in, 1H1, 4.0 (mn, 111, 41T); 3.85 (mn, 2H, 4.2 (in, 2H, 51T); 7.75 IH, H16); 7.75 111, 1121); 2.4 (2 t's, 4H1, H17 H8); 3.3 mn, 411, 119 1110); 4.35 (mn, 1H1, H115) 1. 95 3H1, T-CH 3 Rreparation of 51-HO-A Cj-.CTXAC-01-3' (13A).

The phosphoramidite (14A) was used on the Pharinacia gene assembler to synthesiz~e the 11-mer 5' HO-TpA'pT"pC'pTpTpC'pTpXpApC'--S-3' (10 inMole scale).

The oligomer on the solid support was washed-for minutes with 10t CF 3 COOH in C11 2 C1 2 to affect detritylation and removal of the Boc groups.

Deprotection was completed with aqueous NH 3 (0.88 and the product was purified on a Nucleogen-DEAE 60-7 preparative HPLC column. Oligomer (3A) (90 OD units) eluted from an analytical ion exchange column at 23.5 minutes using the same solvent gradient flowing at inL/min.

Claims (32)

1. 2. The method of Claim 1 wherein said compound is soluble in water at a neutral pH.
2. 3. The method of Claim 1 wherein said metal complex is attached to said compound at any location where chemical derivatization is possible.
3. 4. The method of Claim 1 wherein said metal is selected from the group consisting of copper, zinc, cobalt, nickel, palladium, lead, iridium, manganese, iron, molybdenum, vanadium, ruthenium, bismuth, magnesium, uranium, rhodium and the Lanthanide metals. The method of Claim 1 wherein said metal is selected from the group consisting of copper, zinc and cobalt. C, C C 9 5 30 1 tL# Lu
4. 6. The method of Claim 1 wherein the ligand of said metal complex is selected from the group of compounds consisting of 2,2':6',2"-terpyridine; 2,2'-bipyridine;
5. 7-(N-methyl)-2, 12-dimethyl-3, 7, 11, 17- tetraazabicyclo[11.3.1]heptadeca-1 2, 11,13, 2,12-dimethyl-3, 7, 11, 17-tetraazabicyclo [11.3.1]heptadeca-i 2, 11, 13, 2,10-dimethyl-3, 6, 9, 12-tetraazabicyclo[9.3.1]pentadeca-I 10, 12, 15-pentaene; N-phosphonomethyliminodiacetic acid and 4,4'-dimethyl-2, 2'-bipyridine. -39- 7. The method of Claim 1 wherein said metal complex is selected from the group of complexes formed by reacting 2,21:61, 2"-terpyxidine and copper 2,2'- bipyridine and copper 4,4'-dimethyl-2, 2'-bipyrLidine and copper 7-(N-methyl)-2,
6. 12-dimethyl-3, 7, 11f .:7-tetraazabicyclo Il.3.1]heptadeca-1 2, 11, 13, and zinc 2, 12-dimethyl-3, 7, 11, 17- tetraazabicyclor1l.3.1heptadeca-I 2, 11, 13, pentaene and zinc and 2,10-dimethyl-3, 6, 9, 12- tetraazabicyclo[9.l]pentadeca-I (15) 2, 10, 12, pentaene and copper said complexes may have ancillary ligands selected from the group consisting of chloride, hydroxid~e, water, bromide, iodide, perchiorate, nitrate, sulfate, phosphines, phosphites, and other mono- and is bidentate ligan-ds. 8. A compound consisting of a metal complex covalently linked at any location where chemical derivatization is possible to a nucleoside, nucleotide or oligodeoxynucleotide, which is effective under non- oxidati,,e, physiologically relevant conditions for producing hycdulysis of RNA but ineffective for producing hydrolysis of DNA. 9. The compound of Claim 8 wherein said metal **0complex is covalently linked to a nucleoside or ixucleotide. 10. The compound of Claim 8 which exhibits sequence-directed hydrolysis of ?'TA wherein said metal complex is covalently linked to an oligodeoxynucl3otide. 11. The compound of Claim 8 wherein said metal complex is selected froil the group of complexes defined in 3 0 Claim 7. 12. The compound of Claim 8 wherein said metal complex is linked, off the C-5 position )f uracil in a 2'- deoxy-uridine nucleoside or nucleotide; the C-4 or position of cytosine in a 2'-deoxy-cytidine nucleoside or nucleotide; the N-6 position of adenine in a 2'-deoxy- zc,.-osine nucleoside or nucleotide; or the N-2 or 0-6 p,7o.ition of guanine ir.; a 2'-deoxy-guanosine nucleoside or nucleoti~la.
7. 13. The compound of Claim 12 wherein said metal complex is selected from the group of complexes defined in Claim 7.
8. 14. The compound of Claim 8 wherein the metal in said metal co,,nplex is selected from the group consisting of copper, zinc and cobalt.
9. 15. The compound of Claim 8 wherein the ligand in said metal complex is selected from the group consisting of 2,2' -bipyridine and substituted 2,2' -bipyridines.
10. 16. The compound of claim 8 wherein the ligand in said m'~tal complex is selected from the group consisting of "920 2,2':6',2"-terpyridine and suh-'stitUted terpyridines. .9 17. The complex of bipyridinfl4ylloxobutyl-amino~ethyl~amino]-3- 25 18. The complex of 31-[4-[4'-methyl[2,2'- bipyridin)-4-yljbutyl-phosphate--2 '-deoxy-thymidine ammonium salt and copper (II). -41-
11. 19. The complex of 5'-[4-[4'-methyl[2,2'- bipyridin]-4-yl]butyl-phosphate]-2'-deoxy-thymidine triethylammonium salt and copper (II). The method of covalently linking a metal to a nucleoside, nucleotide or oligodeoxynucleotide by reacting said nucleoside, nucleotide or oligodeoxynucleotide and metal complex under conditions to covalently link said metal complex to said nucleoside, nucleotide or oligodeoxy- nucleotide at any location where chemical derivatization is possible, said metal complex being effective under non- oxidative, physiologically relevant conditions for producing hydrolysis of RNA but ineffective for producing hydrolysis of DNA.
12. 21. The method for manufacturing a compound as defined in Claim 1 by reacting a nucleoside, nucleotide or oligodeoxynucleotide and an organic ligand which when complexed with a metal ion is effective for producing under non-oxidative, ph siologically relevant conditions hydrolysis of RNA, to covalently link said organic ligand to said nucleoside, nucleotide or oligodeoxynucleotide at any location where chemical derivatisation is possible and, then, reacting the resulting compound with a metal ion which under non-oxidative, physiologically relevant conditions is effective for producing hydrolysis of RNA but ineffective for producing hydrolysis of DNA, so as to attach said metal ion to said organic ligand.
13. 22. The method of Claim 20 wherein a metal ion which is effective under non-oxidative, physiologically :.relevant conditions for producing hydrolysis of RNA but ineffective or producing hydrolysis of DNA, is first '0 reacted with said ligand to form a complex which is then r::eacted with said nucleoside, nucleotide or oligodeoxy- nucleotide to covalently link said complex at any location where chemical derivatization is possible. -42-
14. 23. a process for the hydrolysis of RNA under non-oxidative, physiologically relevant conditions the improvement which consists of contacting said RNA with a metal complex covalently linked to a nucleoside, nucleotide or oligodeoxynucleotide to provide sequence-dirocted hydrolysis.
15. 24. The process of Claim 23 wherein said metal complex is covalently linked to an oligodeoxynucleotide. The process of Claim 23 wherein said metal complex is attached to said oligodeoxynucleotide at any location where chemical derivatization is possible.
16. 26. The process of Claim 23 wherein said metal is selected from the group defined in Claim 4.
17. 27. The process of Claim 23 wherein said metal is selected from the group consisting of copper, zinc and cobalt.
18. 28. The process of Claim 23 wherein the ligand of said metal complex is selected from the group of compounds defined in Claim 6. 1
19. 29. The process of Claim 23 wherein said metal complex is selected from the group of complexes defined in Claim 7. 94
20. 30. The method of cleaving RNA with a compound selected from the group consisting of nucleosides, nucleotides and oligodeoxynucleotides having covalently linked thereto two or more imidazole groups. i S f I vl cy -43-
21. 31. The method of cleaving RNA with a combination of two compounds selected from the group consisting of nucleosides, nucleotides and oligodeoxynucleotides each having.one or more imidazole groups attached thereto, which combination is effective under physiologically relevant conditions, for producing cleavage of RNA.
22. 32. The method of cleaving RNA with a compound selected from the group consisting of nucleosides, nucleotides and oligodeoxynucleotides having attached thereto at least one imidazole group in the presence of a solution containing at least one imidazole group.
23. 33. The method of any one of claims 30 to 32 wherein said attached imidazole groups are attached to said compounds at any location where chemical derivatization is possible as long as the essential properties of the imidazoles remain intact, so that they may function as either acids, bases or both, in either the Lewis or Bronsted definitions of the terms.
24. 34. A compound containing two or more imidazole groups effective for cleaving RNA under physiologically relevant conditions, covalently linked to a nucleoside, nucleotide or oligodeoxynucleotide at any location where chemical derivatization is possible as long as the essential properties of the imidazoles remain intact, so that they may function as either acids, bases or both, in either the Lewi. or Bronsted definitions of the terms.
25. 35. A compound of Claim 34 which exhibits sequence-directed cleavage of RNA wherein said imidazole groups are linked to an oligodeoxynucleotide. v:; -44-
26. 36. A compound of Claim 34 wherein said imidazole groups are juxtaposed in an appropriate manner to enhance cleavage of RNA.
27. 37. The compound of 3 -[lH-imidazol-4-yl]propyl]amino]-l-oxo-3-[1H-imidazole-4- yl]propyl]amino]ethyl]amino]-3-oxopropyl]-2'-deoxyuridine.
28. 38. A combination of compounds containing one or more imidazole groups effective for cleaving RNA under physiologically relevant conditions, covalently linked to two or more nucleosides, nucleotides cr oligodeoxy- nucleotides at any location where chemical derivatization is possible as long as the essential properties of the imidazoles remain intact, so that they may function as either acids, bases or both, in either the Lewis or Bronsted definitions of the terms.
29. 39. The combination of compounds of Claim 38 wherein said imidazole groups are juxtaposed in an appropriate manner to enhance cleavage of RNA. The method of covalently linking two or more imidazole groups effective for RNA cleavage under physiologically relevant conditions to a nucleoside, nucleotide or oligodeoxynucleotide by reacting said nucleoside, nucleotide or oligodeoxynucltotide and imidazole groups under conditions to covalently link said imidazole groups to said nucleoside, nucleotide or oligodeoxynucleotide at any location where chemical deru-vizazation is possible as long as the reactivity of the imidazole is not prevented from further chemical reaction. *.4Q 0 too 0. to 4 4*44 :0.40 o.so '&C Sj r
30. 41. In a process for the cleavage of RNA under physiologically relevant conditions the improvement which consists of contacting said RNA with a compound selected from the group containing two or more imidazole groups covalently linked to an oligodeoxynucleotide or two or more oligodeoxynucleotide having one or more imidazole groups covalently linked thereto to provide sequence-directed cleavage.
31. 42. In a process for the cleavage of RNA under physiologically relevant conditions the improvement which consists of contacting said RNA with a compound selected from the group containing one or more imidazole groups covalently linked to an oligodeoxynucleotide in the presence of a solution containing an imidazole group.
32. 43. The process of Claim 41 wherein said imidazole groups are attached to said oligodeoxynucleotide at any location where chemical derivatization is possible so long as the essential properties of the imidazoles remain intact, so that they may function as either acids, bases or 20 both, in either the Lewis or Bronsted definitions of the terms. I DATED this 8th day of September, 1993 te o MONSANTO COMPANY, By its Patent Attorneys, E .L WELLINGTON CO., I -7 I,, S. Wellington' A/RR/2185/13 Ii