Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H23/00—Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12

Definitions

• 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 agents and to RNA cleavage generally and particularly to two or more imidazoles covalently linked to oligodeoxynucleotides as sequence-directed RNA cleavage agents.
• RNA ribonucleic acids
• t tramine complexes of Co(III) are capable of promoting the hydrolysis of adenosine 3',5'-monophosphate (cAMP) (J. Chin et al, Can . J. Chem . 1987, 65, 1882-1884) and adenosine monophosphate (AMP) (J. Chin et al, J. Am. Chem. Soc. 1989, 111, 4103-4105).
• cAMP adenosine 3',5'-monophosphate
• AMP adenosine monophosphate
• it is known that many divalent cations are capable of catalyzing the hydrolysis of RNA (J. J. Butzow et al, Biochemistry 1971, 10, 2016-2027 and J. J. Butzow et al, Nature 1975, 254, 358-359).
• zinc ion in the presence of imidazole buffers has been sliown to catalyze the hydrolysis of the RNA dimer 3'
• 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 15, 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).
• investigations into phosphate ester hydrolysis have been carried out using imidazole and imidazole derivatives as models for histidine. Such reported studies have generally
• 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 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 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
• analogues are convenient models for biological substrates, they are not accurate models (Menger, F. M. and Ladika, M., J. Am. Chem . Soc. 1987, 109, 3145.)
• 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 endonucleases, 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
• RNA hydrolysis agents presently are available only at a high cost due to limitations of very low production volumes through molecular biology techniques.
• 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-phenanthrolinecopper-oligodeoxynucleotide conjugate employed is itself degraded oxidativelj under the conditions of oxidative RNA cleavage (the r ⁇ te of oxidative cleavage by the
• 1,10-phenanthrolinecopper system is similar for both RNA and DNA) .
• P. G. Schultz and coworkers in a series of articles D. 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 .
• 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
• 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, 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 the 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 (i.e. to 0oC).
• RNA hydrolysis and cleavage are necessary to provide a basis for catalytic antisense drug development.
• 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, nucleotides and oligodeoxynucleotides containing two or more imidazole groups attached via an appropriate linker, to the cleavage of RNA by a
• oligodeoxynucleotides each containing at least one imidazole group attached via an appropriate linker
• imidazole group attached via an appropriate linker to a nucleoside, nucleotide 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.
• 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 Br ⁇ nsted
• a first aspect of this invention 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, nucleotides and oligodeoxynucleotides useful for promoting RNA cleavage.
• the oligodeoxynucleotide provides molecular recognition via Watson Crick base pairing to the target RNA sequence.
• 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 (l) the discovery of metal complexes which are effective for the hydrolysis of RNA, (2) the discovery that molecules containing two or more imidazole groups show great enhancement over mono-imidazole species for the cleavage of RNA, (3) the preparation of conjugates which retain RNA cleavage behavior, (4) the preparation of oligodeoxynucleotide conjugates effective for the sequence-directed
• FIG. 2 there is shown the titration of 3'-[4-[4'-methyl(2,2'-bipyridin)-4-yl]butyl-phosphate]-2'-deoxy-thymidine ammonium salt (5) with CuCl 2 forming 3'-[4-[4'-methyl(2,2'-bipyridin)-4-yl]butyl-phosphate]-2'-deoxy-thymidine ammonium salt copper(II) (6).
• This Figure depicts Applicants' Example IV and demonstrates the formation of a metal complex nucleotide conjugate in accordance with Applicants' ihyehtion.
• FIG. 3 there is shown the hydrolytic cleavage of RNA [poly(A) 12-18 ] by compound (6).
• This Figure depicts Applicants' Example V and demonstrates that a metal complex linked to the 3' position of a nucleotide is capable of hydrolyzing RNA.
• FIG 4 there is shown the hydrolytic cleavage of RNA [poly(A) 12-18 ] by 5'-[4-[4'-methyl(2,2'- bipyridin)-4-yl]butyl-phosphate]-2'-deoxy-thymidine triethylammonium salt copper(II) (12).
• 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.
• Figure depicts Applicants' Example XI and demonstrates that a metal complex linked to the base portion of a nucleoside is capable of hydrolyzing RNA.
• FIG. 6 there is shown the reaction of the Cu(bpy) 2+ complex with both DNA [poly(dA) 12-18 ] and RNA [p-oly(A) 12-18 ].
• 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;
• FIG. 7 there is shown the densitometry results of polyacrylamide gel electrophoresis analysis of the sequence-directed hydrolysis of tRNA Tyr by the oligodeoxynucleotide-Cu(bpy) 2+ conjugate (32).
• This Figure depicts Applicants' Example XV showing Densitometry scans of the polyacrylamide gel of the reaction of (32) with tRNA Tyr after 17 hours under the conditions described in Example XV and of the control reaction.
• FIG. 9 there is shown the autoradiograph which depicts the cleavage of 32 P labeled RNA by compound (5A) and that compound (6 ⁇ ) is ineffective at cleaving RNA.
• 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 1 depicts the synthesis of compound (5) as described in Applicants' Example III.
• Scheme 5 depicts the synthesis of compound (30) as described in Applicants' Example XIV.
• Scheme 6 depicts the sequence-directed cleavage of tRNA Tyr by compound (32) as described in Applicants' Example XV.
• Scheme 7 depicts the synthesis of compound (5A) 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' Example XVIII.
• the hydrolytically effective oligodeoxynucleotide conjugates of this invention are comprised of a desired organic molecule, herein referred 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 nucleosides, nucleotides or oligodeoxynucleotides.
• Applicants' invention is based on metal complexes which are effective for RNA hydrolysis, and the preparation of such metal complexes covalently linked to nucleosides, nucleotides 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.
• Agents as used herein means Applicants' synthetic RNA hydrolysis compounds comprising a metal and a ligand, 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, (2) increased chemical stability over ribozymes, (3) ease of
• 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
• Ribonuclease S is a noncovalent complex comprised of the S-protein and S-peptide derived from ribonuclease A. This complex is subject to
• 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. This 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 enzyme-based systems was artificially increased by lowering the temperature below physiologically relevant values (i.e. to 0oC).
• RNA 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.
• 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
• oligodeoxynucleotide conjugates will not cleave their own oligodeoxy- nucleotide components at an appreciable rate. See Applicants' Example II below.
• oligodeoxynucleotides used herein includes oligodeoxynucleotides and oligodeoxynucleotide analogs that are. effective at molecular recognition by, for example, Watson-Crick or Hoogsteen base-pairing.
• 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, 1989, Chapter 4 and references therein
• compounds with sulfur-containing internucleotide 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).
• oligodeoxynucleotide analogs which may be suitable include those with internucleotide linkages such as carbonate, acetate, carbamate, dialkyl and diarylsilyl groups.
• the metal complexes which hydrolyze RNA may contain hydroxyl or aquo ligands or both. These active forms may be derived in a
• 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,
• RNA under physiologically relevant conditions 7.1 pH and 37oC.
• Applicants' invention is based on (1) the use of two or more imidazole groups for RNA cleavage, and (2) the preparation of conjugates
• imidazole groups covalently linked to nucleosides, nucleotides and oligodeoxynucleotides.
• a mixture of adenylic acid oligomers 12 to 18 nucleotides in length [poly(A) 12-18 ] is used as the assay substrate.
• Ion exchange HPLC is used to resolve the individual cleavage products from the substrate
• 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.
• 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.
• the selected ligand may first be covalently linked to the desired nucleoside or nucleotide and then the selected metal ion attached to the ligand.
• 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 selected ligand may first be covalently linked to the desired oligodeoxynucleotide and then the selected metal ion attached to the ligand.
• the intact selected metal complex may be covalently linked to the oligodeoxynucleotide.
• the ligand or metal complex can be covalently linked to the oligodeoxynucleotide at any location.
• nucleosides nucleotides and oligodeoxynucleotides.
• This Example shows how metal complexes and other compounds are screened for RNA hydrolysis activity.
• RNA 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) 12-18 ] was prepared by dissolving 25 units of the DNA in l.OmL of 20mM HEPES buffer pH - 7.1.
• the reaction mixture contained in a total volume of 1.5mL, 63 ⁇ M of the DNA, 157 ⁇ M bipyridine, 157 ⁇ M CuCl 2 and 20mM 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+18 ].
• 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-phenathroline-copper(II) at similar rates (C. B. Chen et 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.
• This Example shows the attachment of bipyridyl ligand (bpy) to the 3' position of 2'-deoxy-thymidine nucleotide as outline in Scheme 1.
• nucleoside 3 '-[4-[4'-methyl(2,2'-bipyridin)-4-yl]butyl-phosphate]-2'-deoxy-thymidine ammonium salt (5) (0.192 gm., 0.354 mmol, 82%).
• compound (6) was formed under the conditions set forth in Example IV.
• time zero a lOO ⁇ L aliquot of the reaction mixture was removed and immediately analyzed by Applicants' HPLC Assay.
• the reaction mixture was incubated at 37oC for 48 hours after which time a second aliquot was removed and assayed. It was found that the RNA substrate was clearly hydrolyzed by compound (6) ( Figure 3).
• This Example shows the attachment of bipyridyl ligand (bpy) to the 5' position of 2'-deoxy-thymidine nucleotide as outlined in Scheme 2.
• phosphoramidite (8) (0.101 gm., 0.25 mmol) and tetrazole in lmL of THF was stirred at room temperature for 10 minutes.
• 3'-O-acetyl-2'-deoxythymidine (7) (0.071 gm., 0.25 mmol) dissolved in CH 2 Cl 2 (lmL) was added to the reaction mixture and the solution was left stirring for 60 minutes. The mixture was then filtered to remove tetrazole which precipitated out.
• This Example shows the titration of compound (11) prepared in Example VI with CuCl 2 to form 5'-[4-[4'-methyl(2,2'-bipyridin)-4-yl]-butyl-phosphate]-2'-deoxythymidine triethylammonium salt copper(II) (12).
• Example II The procedure described in Example II was followed. Changes in visible spectrum, similar to those shown in Figure 3, characteristic of coordination of copper(II) to bipyridine were observed. Titration of thymidine-5'-monophosphate showed no changes in the visible spectrum over the range 240-380nm.
• 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.
• This Example shows the titration of compound (16) with CuCl 2 to form 5-[3-[[2-[[4-[4'-methyl[2,2'-bipyridin]-4'-yl]-1-oxobutyl]amino]ethyl]amino]-3-oxopropyl]-2'-deoxy-uridine copper(II) (17).
• Example III The procedure described in Example III was followed. 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 showed no changes in the visible spectrum over the range 240-380nm.
• 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.
• trpy terpyridine
• the mixture was poured over 10 ml brine and the aqueous layer was extracted with CH 2 Cl 2 .
• the extracts were dried over MgSO 4 and evaporated to dryness to yield the crude acetal.
• the acetal was hydrolyzed with 1M HCl (10mL) by heating to 50-60oC for 2 hours. The solution was then neutralized with aqueous NaHCO 3 and extracted with
• Compound (23) can be attached to the 5' position of 2'-deoxy-thymidine by literature procedures (see B. C. F. Chu et al, DNA 1985, 4, 327-331).
• This Example shows a variety of nucleosides and nucleotides which have groups appended on the 3' and 5' position of 2'-deoxy-thymidine and 5- position uracil in 2'-deoxy-uridine and which are not active at hydrolyzing RNA [poly(A) 12-18 ] under the conditions of the HPLC assay (Table 2).
• This Example shows the attachment of bipyridine ligand to a 14mer oligodeoxynucleotide (30) as shown in Scheme 5.
• oligodeoxynucleotide-bipyridine conjugate (31) 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.
• This Example shows a sequence-directed cleavage of tRNA Tyr by an oligodeoxynucleotide-bipyridine
• a 10l ⁇ M 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 25.9 ⁇ M stock solution of the tRNA Tyr substrate was prepared by dissolving 10 Units of tRNA Tyr in 500 ⁇ L of 20 mM HEPES buffer having a pH of 7.1.
• the cleavage reaction contained in a total of 600 ⁇ L, 1.29 ⁇ M tRNA Tyr , 12.9 ⁇ M Cu(trpy) 2+ , 227 ⁇ M Cu(SO 4 ), 6.4 ⁇ M compound (31), 50mM NaCl and 50mM HEPES buffer having a pH of 7.8. Initially the tRNA Tyr , compound (31), NaCl and buffer were combined and heated to 65*C for 4 min. in a water bath. The reaction was removed and
• 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 (32).
• the reaction was heated at 37oC and 100 ⁇ L 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.
• This example shows the synthesis of the diimidazole containing nucleoside (5A) , 5-[3-[[2-[[2-[[2-amino]-1-oxo-3-[1H-imidazol-4-yl]propyl]amino]-1-oxo-3-[1H-imidazol-4-yl]propyl] amino]ethyl]amino]-3oxopropyl]-2'-deoxy-uridine, as outlined in Scheme 7.
• the synthesis of compound (IA) has been previously described (Dervan et al., Proc. Natnl . Acad. Sci . USA 1985, 82, 968).
• the nucleoside 5'-O-DMT-5-[3-[(2-aminoethyl)-amino]-3-oxopropyl]-2'-deoxy-uridine (IA) (1.288 g, 2.0 mmol) was dissolved in dry dichloromethane (10 mL) and was cooled to 0oC in an ice bath.
• Fmoc-L-His(Tr)-O-pfp (3.16 g, 3.0 mmol) 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
• the nucleoside (2A) (1.94 g, 1.56 mmol) dissolved in CH 2 Cl 2 (10 mL) was treated for 3 hours at room temperature with Et 2 NH (10 mL) and the mixture was concentrated to a glass. The residue was flash chromatographed on a silica gel column.
• the deprotected impurities were eluted off the column using CH 2 Cl 2 and the nucleoside 5'-O-[Bis(4-methoxyphenyl)phenylmethyl]- 5-[3-[[2-[amino]-3-[1-(triphenylmethyl)-1H-imidazol-4- yl]propyl]amino]ethyl]amino]-3-oxopropyl]-2'-deoxyuridine (3A) (1.43 g, 1.4 mmol, 90%) was eluted off with 80% EtOH in CH 2 Cl 2 .
• the nucleoside (4A) (0.4 g, 0.246mmol) was dissolved in CH 2 Cl 2 (5 ml) and was treated with
• 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-(1H-imidazol-4-yl)-1-oxopropyl]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).
• RNA substrate was generated by runoff transcription with bacteriophage SP6 DNA-dependent RNA polymerase using standard techniques (Maniatis, T.;
• HEPES buffer purchased from Sigma
• Stock solutions of (5A), 4.9 mM, and (6A), 3.1 mM were prepared in 20 mM HEPES buffer.
• the control reaction (#l) contained in a total volume of 25 ⁇ L, 4.5 ⁇ g 32 P labeled RNA which was diluted to volume with HEPES buffer.
• the reaction (#2) with agent (5A) contained in a total volume of.90 ⁇ L, approximately 23 ⁇ g of 32 P labeled RNA and 1 mM compound (5A).
• reaction #2 shows nearly complete cleavage of RNA (Lanes #3 , 4, 5, 6,) while reactions #1 (Lane #1) and #3 (Lane #l and 8) are devoid of RNA cleavage.
• reaction #3 shows no cleavage even with 2 mM total imidazole concentration indicates that incorporating two imidazoles into the same
• imidazole-nucleoside conjugates into di-, tri-, and oligodeoxynucleotides. These techniques may be applied for the incorporation of suitably protected conjugates into oligodeoxynucleotides.
• 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 diastereomers; it was further characterized by FABMS and high resolution mass spectra.
• the 1 H NMR peak assignments of (15A) are given in the experimental section.
• 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 nucleoside analogue.
• HEMS high resolution mass spectra
• the FAB matrix was a saturated solution of Lil in 3-nitrobenzyl alcohol, which is especially useful for acid-labile, protected nucleosides.
• Thin layer chromatography was performed on Baker-Flex Silica gel IB2-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-60, 230-240 mesh). RP HPLC was carried out on a Alltech Econosil C18
• o-Chlorophenyl phosphorodichloridate (0.897 g, 3.66 mmol) was weighed into a two-necked pear-shaped flask and dissolved in acetonitrile (10 mL). 1,2,4-Triazole (0.556 g, 8.052 mmol) and triethylamine (1.02 mL, 7.32 mmol) were added to the reaction vessel, and the mixture stirred at room temperature for 20 minutes. The nucleoside (7A) was dissolved in acetonitrile (10 mL), 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 20 minutes. The reaction was monitored on TLC, and after all of the starting material was consumed, the mixture was quenched with triethylamine (3.06 mL, 21.96 mmol) and water (10 mL) to give a homogeneous solution. The solution was stirred for 10 minutes and then concentrated. The residue was dissolved in dichloromethane (25mL) and washed with sat. NaHCO 3 (25 mL). The aqueous layer was washed with dichloromethane (2 ⁇ 20mL), and the combined organic extracts were dried over MgSO 4 and concentrated to a glass.
• the phosphodiester 8 (0.427 g, 3.36 mmol) was dissolved in dry pyridine (5 mL) and 4-N-3'-O-diacetyl- 2'-deoxycytidine (0.095 g, 3.05 mmol) 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 of moisture. 1-(2- mesitylenesulfonyl)-3-nitro-1,2,4-triazole (0.361 g, 12.2 mmol) was then added to the solution of the two nucleosides and stirred at room temperature for 20-25 minutes. The mix:cure was then quenched with 1 mL saturated solution of NaHCO 3 .
• the fully protected dimer (9A) (0.220 g, 0.15 mmol) was treated with a freshly prepared solution of N 1 , N 1 , N 3 , N 3 -tetramethylguanidine (0.33 M) and o-nitrobenzaldoxime in dry CH 3 CN (1.5 mL). After 3 hours at room temperature the mixture was concentrated 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% CF 3 COOH in
• nucleoside was extracted with water (10 mL), and the aqueous layer was washed with diethyl ether (2 ⁇ 5 mL). The aqueous layer was evaporated to yield the
• nucleoside (10A) (0.110 g, 0.143 mmol, 95% ).
• the sample was purified on a Alltech Econosil C18 preparative RP HPLC column. Retention time for (10A) (250 OD units) was 14.6 minutes on a C18 analytical column using the same linear ternary gradient, flowing at 1.5 mL/min.
• Chloro-N,N-diisopropylamino- ⁇ - cyanoethoxyphosphme (0.158 g, 0.81 mmol) was weighed into an H-shaped Schlenk flask and dissolved in
• acetonitrile (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. The nucleoside (7A) (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 solutions were concentrated to yield a glass. The glass was then chromatographed on a silica gel chromatotron (2000M) .
• 2000M silica gel chromatotron

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