Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
  • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
  • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
  • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
  • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
  • C07D239/52—Two oxygen atoms
  • C07D239/54—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Definitions

• the invention is in the field of polynucleotide analogs lacking furanose rings.
• Oligonucleotides that bind sequence specifically to complementary nucleic acids (i.e. sense strand) by hydrogen bonding so as to inhibit gene expression are commonly referred to as antisense oligonucleotides.
• oligonucleotides bind to target (mRNA) and thereby inhibit translation of the messenger RNA. This antisense principle
• Zamecnik and Stephenson were the first to propose, in 1978, the use of synthetic oligonucleotides for therapeutic purposes (Stephenson, M. L.; and Zamecnik, P. C, Proc. Na tl . Acad. Sci . USA, 1978, 75, 280 and 285).
• the specific inhibition of antisense polynucleotide is based on the specific Watson-Crick base pairing between the heterocyclic bases of the antisense oligonucleotide and of viral nucleic acid. The process of binding of the
• oligonucleotides to a complementary nucleic acid is called hybridization.
• oligonucleotides having a DNA structure The preparation of unmodified oligonucleotides, i.e., oligonucleotides having a DNA structure, has been the center of interest for many research groups in the past decade.
• the synthesis via phosphoramidites according to Caruthers according to Caruthers
• oligonucleotides When normal, i.e., unmodified,
• oligonucleotides are used as antisense oligonucleotides, the problems of instability to nucleases and insufficient membrane penetration have arisen. For antisense oligonucleotides to be able to inhibit translation they must reach the interior of the cell unaltered.
• the properties useful for oligonucleotides to be used for antisense inhibition include: (i) stability of the oligonucleotides towards extra- and intracellular enzymes; (ii) ability to penetrate through the cell membrane; and (iii) ability to hybridize the target DNA or RNA (Agarwal, K. L. et al., Nucleic Acids Res . , 1979, 6, 3009; Agawal, S.
• n is the number of modified diester linkages in the oligomer.
• PNA Acids
• phosphorus atom in either the pentavalent or trivalent oxidation state.
• Specific coupling procedures have been referred to as the phosphite triester (phosphoramidite), the phosphorus diester, and the hydrogen phosphonate procedures.
• hybridization efficiency increased target specificity, stability against nucleases, improved cellular uptake, and assistance in the important terminating events of nucleic acids (e.g. RNase H activity, catalytic cleavage, hybridization arrest, and others).
• RNase H activity e.g. RNase H activity
• catalytic cleavage e.g. RNase H activity
• hybridization arrest e.g. RNase H activity
• carbonate diesters e.g. RNase H activity, catalytic cleavage, hybridization arrest, and others.
• oligonucleotides are found to be enzymatically stable and form base pairing with the complementary sequence. Given the shortcomings of polynucleotides and known polynucleotide analogs, it is of interest to provide new polynucleotide analogs for use in antisense inhibition and other techniques employing oligomers.
• the present invention provides novel oligonucleotides, and structural precursor thereof, which have improved
• novel oligonucleotides of the present invention improved hybridization properties with respect to nucleic acid hybridization targets.
• the oligomers of the present invention are generally characterized as comprising a series of constrained linkers or monomers that is appropriate for binding of heterocyclic bases to a target nucleic acid in a sequence specific manner.
• the constrained linkers described herein, when incorporated into oligomers, may have a force greater than a single hydrogen bond, thereby favoring formation of the binding competent conformation.
• the nucleomonomers of the present invention are generally characterized as moieties or residues that replace the
• nucleotides with an amino acid or a modified amino alcohol residues.
• exemplary monomers and oligomers of the invention are shown in formulae 1 through 41. Incorporation of these monomers described herein into oligonucleotides permits synthesis of compounds with improved properties, these
• improved properties include (i) increased lipophilicity which results from eliminating the charge associated with
• the present invention provides various novel features
• the compounds of the invention are N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
• oligonucleotide analogs in which the furanose ring of a naturally occurring nucleic acid is replaced with an amino acid or a modified amino alcohol residue.
• Some embodiments of the novel compounds of the invention are particularly useful for the antisense control of gene expression.
• the compounds of the invention may also be used as nucleic acid
• hybridization probes or as primers are hybridization probes or as primers.
• Another aspect of the invention is to provide monomeric precursors of the oligonucleotide analogs of the invention. These monomeric precursors may be used to synthesize the subject polynucleotide analogs.
• Another aspect of the invention is to provide
• Yet another aspect of the invention is to provide methods for treating or preventing diseases, particularly viral infections and cell growth disorders.
• the subject disease treatment methods comprise the step of administering an effective amount of the subject polynucleotide analogs for use as antisense inhibitors.
• FIGS 1 through 25 are depictions of chemical reaction sequences usable for synthesizing monomers
• Figure 1 shows the synthesis of L-serinol coupled thymine monomer phosphoramidite with a -CH 2 -CO- linkage between thymine and serinol.
• Figure 2 shows the synthesis of L-serinol coupled thymine monomer phosphoramidite with a -CH 2 -CH 2 - linkage between thymine and serinol.
• Figure 3 and 4 depicts the synthesis of substituted L- serinol coupled thymine monomer phosphoramidites with a -CH 2 - CO- linkage between thymine and serinol.
• Figure 5 shows the synthesis of T-T dimer with 5 atom long inter nucleotide linkage having hydroxylamine in the middle of the internucleotide linkage with a -CH 2 -CO- linkage between thymine and serinol.
• Figure 6 depicts the synthesis of thymine monomer
• phosphoramidite in which thymine is connected to an N-ethylhydroxylamine through a -CH 2 -CO- linkage.
• Figure 7 shows the synthesis of L-serinol coupled thymine monomer phosphoramidite in which the NH 2 group of L-serine is connected to 2-hydroxyacetyl group and the hydroxy function is blocked with DMT group. This building block is used for 2'-5' connection. This figure also depicts the synthesis of thymine monomer in which the NH 2 group of L-serine is connected to a
• Figure 8 shows the synthesis T-T dimer having a
• hydroxamate backbone with 2'-5' linkage In this dimer one building block is made from L-aspartic acid and thymine and the other is from L-serine and thymine. This dimer lacks amide bond between in the backbone.
• Figure 10 shows the synthesis of L-serinol-b-alanine coupled thymine monomer phosphoramidite in which ⁇ -alanine links thymine and serinol.
• Figure 11 shows the synthesis of L-serinol-akylamine coupled thymine monomer phosphoramidite with alkyamine links thymine and serinol.
• FIG. 12 depicts the synthesis T-T dimer having
• the dimer is made from two L-aspartic acid units and two thymine units with an acetyl linker between thymine and aspartic acid.
• FIG. 13 depicts the synthesis T-T dimer having
• the dimer is made from two L-aspartic acid units and two thymine units with an ethyl linker between thymine and aspartic acid.
• Figure 14 shows the synthesis of N-hydroxyamino acid coupled thymine building block.
• Figure 15 shows the synthesis of L-aspartic acid coupled thymine building block with an N-hydroxylamine linker between thymine and aspartic acid.
• Figure 16 depicts the synthesis T-T dimer having a hydroxamate backbone with 4'-5' linkage. In this, the carboxylic acid group is coupled to thymine building block through an N-hydroxylamine linker.
• Figure 17 depicts the synthesis thymidineacetic acid substituted N-hydroxyamino acid building block 150 and its analogue 149. These monomer building blocks are useful to create nucleic acid with hydroxamate backbones.
• Figure 18 shows the synthesis of thymidineacetic acid substituted hydroxylamine containing amino acid building blocks 157 and 158. These monomers are useful to design nucleic acid having amide backbone with hydroxylamine
• Figure 19 shows the synthesis of L-serinol coupled thymidine building block 166 having a hydroxylamine moiety between thymine and serinol. This building block is useful to devise nucleic acid of 4'-5' linkages.
• Figure 20 depicts the synthesis of glutamic acid-glycine coupled Thymidine monomer 174. This monomer building block is useful to generate nucleic acid with amide backbones and 2'-5' linkages.
• Figure 21 shows the synthesis of glycinol-glycine coupled thymidine building block 181 and 182 having a hydroxylamine moiety between thymine and glycinol. These building blocks are useful to prepare nucleic acid of 2'-5' linkages.
• Figures 22 through 25 indicate the synthesis of ribose amino acid coupled thymidine building blocks 191, 199 and 207. These building blocks are useful to prepare oligonucleotides having ribose-amide backbone.
• Figure 25 depicts the solid phase synthesis of
• oligonucleotide 211 having ribose-amide backbone.
• Figure 23 shows the synthesis of 1-O-(4,4'-Dimethoxy- trityl)-2-[amino(thyminylacetyl)]-L-propan-3-O-(N,N-diisopropy 1)- ⁇ -cyanoethylphosphoramidite.
• Figure 24 shows the synthesis of 1-O-(4,4'-Dimethoxytrityl)-2-[amino(thyminylacetyl)]-D-propan-3-O-(N,N-diisopropyl)- ⁇ -cyanoethylphosphoramidite.
• Figure 25 shows the synthesis of 2-[( ⁇ -(4,4'-Dimethoxy ⁇ trityl)-O-acetyl)amino]-3-thyminyl-L-propan-1-O-(N,N-diisopropyl)- ⁇ -cyanoethylphosphoramidite.
• Figure 26 shows the synthesis of N-(Thyminylacetyl)-N- [[(2-isobutyryl)oxy]ethyl]-O-benzylhydroxylamine.
• Figure 27 shows the synthesis of (2R, 4S)-1-(tert-Butyloxycarbonyl)-2-[N 3 -benzoyl(thymin-1-yl)]methyl-4-phthalim ido-pyrrolidine.
• antisense therapy refers to
• binding may be by conventional base pair complementarily, or the binding may through other mechanisms, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.
• antisense refers to a range of techniques generally employed under this
• Oligomer or “Oligonucleotide” are used interchangeably and include naturally occurring compounds such as RNA and DNA, as well as synthetic analogs thereof,
• oligomer and “oligonucleotide” refer to both DNA/RNA and to synthetic analogs thereof.
• oligomer refers to compounds comprising two or more
• nucleomonomers covalently attached to each other by a
• an oligomer can have as few as two covalently linked nucleomonomers ( a dimer ) or may be significantly longer. Oligomers can be binding competent and, thus, can base pair with single-stranded or doublestranded nucleic acid sequences. Oligomers (e.g. dimers hexamers) are also useful as synthons for longer oligomers as described herein. Oligomers may contain abasic sites and pseudonucleosides. The Oligomers includes oligonucleotides,
• oligonucleosides polydeoxyribo-nucleotides (containing 2'-deoxy-D-ribose or modified forms thereof), i.e., DNA, polyribonucleotides ( containing D-ribose or modified forms thereof), i.e., RNA, and any other type of polynucleotide which is an N-glycoside or C-glycoside of purine or pyrimidine base, or modified purine or pyrimidine base.
• Oligomer as used herein is also intended to include compounds where adjacent nucleomonomers are linked via hydroxamate linkages.
• oligomers such as the furanose ring and/or the phosphodiester linkage can be replaced with any suitable functionally equivalent element.
• the term "Oligomer” is intended to include any structure that serves as a chassis or support for the bases wherein the chassis permits binding to target nucleic acids in a sequence-dependent manner.
• Oligomers that are currently known can be defined into four groups that can be characterized as having (i) phosphodiester or phosphodiester analog (phosphorothiaoate, methyl-phosphonate, etc) linkages, (ii) substitute linkages that contain a non-phosphorous isostere (riboacetal, formacetal, carbamate, etc), (iii) morpholino residues, carbocyclic residues or other furanose sugars, such as arabinose, or a hexose in place of ribose or deoxyribose and (iv)
• nucleomonomers linked via amide bonds or acyclic nucleomonomers linked via amide bonds or acyclic
• nucleomonomers linked via any suitable substitute linkage are nucleomonomers linked via any suitable substitute linkage.
• Nucleomonomer refers to a moiety comprising (1) a base covalently linked to (2) a second moiety. Nucleomonomers include nucleosides, nucleotides or bases connected to an amino alcohol. Nucleomonomers can be linked to form oligomers that bind to target or complementary base sequences of nucleic acids in a sequence specific manner.
• a "second moiety" as used herein refers to a compound linked to a Nucleomonomer, and includes an amino acid/amino alcohol moiety, usually serinol, aspartic acid, glutamic acid, glycine, and those species which contain modifications of the amino acid moiety, for example, wherein one or more of the hydrogen is replaced with other functionality (see formulae 24-41), or one carboxylic acid is functionalized to an alcohol, amines, thiols, hydroxylamines, and the like.
• Nucleomonomers as defined herein are also intended to include a base linked to an amino acid or amino alcohol and/or amino acid/alcohol analog having a free carboxyl/hydroxyl group and/or a free amino group and/or protected forms thereof.
• nucleoside refers to an amino acid and amino alcohol derivative thereof, as described further below, carrying a purine, pyrimidine, or analogous forms thereof, as defined below, but lacking a linking moiety such as a phosphodiester analog or a modified internucleoside linkage.
• 5' nucleoside is meant the nucleoside which provides the 5' carbon coupling point to the linker.
• the "5"' end of the linker couples to the 5' nucleoside.
• the "3'" end of the linker joins to the 3' position on the next nucleoside.
• a modified nucleoside which does not precisely include a 3' and/or a 5' carbon, it is understood by the person skilled in the art that this "3'" and “5'” terminology to describe strand polarity used by analogy to DNA and RNA.
• nucleoside refers to a base covalently attached to an amino alcohol/ amino acid analog and which contain a linker between base and the amino acid/amino alcohol.
• nucleoside normally includes
• ribonucleosides deoxyribonucleosides, or any other nucleoside which is an N-glycoside or C-glycoside of a base.
• Base refers to a wide variety of nucleoside base, including purine and pyrimidine
• Purines include adenine, guanine and xanthine and exemplary purine analogs include 8-oxo-N 6 -methyladenine and 7-deazaxanthine.
• Pyrimidines include uracil and cytosine and their analogs such as 5-methylcytosine, 5-(1-propynyluracil),
• Bases when joined to a suitable molecular framework, e.g. a phophodiester backbone, are capable of entering into a base pairing relationship that occur in double-stranded DNA or other double-stranded nucleic acids of similar structure.
• Bases may also be capable of entering into a base pairing relationship in a triple helix nucleic acid.
• Sud Modification refers to any amino acid or amino alcohol moiety other than 2'-deoxyribose.
• Amino Acids/Alcohol refers to any natural amino acids and alcohols of both “R' and "S” isomers.
• Nucleoside Linkages refers to the linkage that exists within the monomer.
• Linkage refers to the moiety that is used to connect the base with amino acid/amino alcohol and derivatives thereof.
• Internucleotide Linkages refers to a phophodiester moiety (-O-P(O) (O)-O-) or any other
• Substitute Linkages refers to any analog of the native group or any suitable moiety that covalently couples adjacent nucleomonomers. Substitute linkages include phosphodiester analogs, e.g. such as
• Substitute linkages include the nonphosphorous containing linkages (2', 5' linkages, 3', 5' linkages and 4', 5' linkages) of the invention.
• Crosslinking moiety refers to a group or moiety in an oligomer that forms a covalent bond with a target nucleic acid.
• Crosslinking moieties include covalent binding species that covalently link an oligomer to target nucleic acids either spontaneously (e.g. N 4 ,N 4 -ethanocytosine) or via photoactivation (e.g. psoralen) and the like.
• Blocking Groups refers to a substituent other than H that is covalently coupled to
• oligomers or nucleomonomers either as a protecting group, a coupling group for synthesis, OPO 3-2 , or other conventional conjugate such as a solid support, label, antibody, monoclonal antibody or fragment thereof and the like.
• OPO 3-2 a coupling group for synthesis
• other conventional conjugate such as a solid support, label, antibody, monoclonal antibody or fragment thereof and the like.
• blocking group is not intended to be construed solely as a protecting group, according to slang terminology, but is meant also to include, for example, coupling groups such as a H-phosphonate or a phosphoramidite.
• protecting group refers to any group capable of protecting the O-atom, S-atom or N-atom to which it is attached from participating in a reaction or bonding.
• Such protecting groups for N-atoms on a base moiety in a Nucleomonomer and their introduction are conventionally known in the art.
• suitable protecting groups include: diisobutylformamidine, benzoyl, silyl and the like.
• Suitable protecting groups for O-atoms and S-atoms are, for example, DMT, MMT, FMOC or esters.
• Protecting groups as used herein includes any group capable of preventing the O-atom, S-atom, or N-atom to which it is attached from
• protecting groups for O-, S-, and N-atoms in nucleomonomers are described and the methods for their introduction are conventionally known in the art.
• Protecting groups also include any group capable of preventing reactions and bonding at carboxylic acids, thiols and the like.
• Coupling group refers to any group suitable for generating a linkages or substitute linkage between nucleomonomers such as a hydrogen phosphonate and a phosphoramidite.
• Conjugate refers to any group attached to the oligomer at a terminal end or within the oligomer itself.
• Conjugates include solid supports, such as silica gel, controlled pore glass and polystyrene; labels, such as fluorescent, chemiluminescent, radioactive atoms or molecules, enzymatic moieties and
• oligomer transport agents such as
• conjugate moities include O-cholesterol, polyethylene glycol (PEG), amino acids, intercalators,
• lipids As used herein refers to a structural unit within an oligonucleotide analog of the invention.
• Transfection refers to any method that is suitable for enhanced delivery of oligomers into cells.
• Subject as used herein refers to a plant or animal, including mammal, particularly a human.
• the oligonucleotides may be covalently linked to various moieties such as intercalators, substances which interact specially with the minor groove of the DNA double helix and other arbitrarily chosen conjugates, such as labels
• intercalators such as acridine can be linked through an R-CH 2 - attached through any available -OH or SH, e.g.., at the terminal 5' position of RNA or DNA, the 2' position of RNA, or an OH or SH engineered into the 5 position of
• pyrimidines e.g., instead of the 5 methyl of cytosine, a derivatized form which contains -CH 2 CH 2 CH 2 OH or -CH 2 CH 2 CH 2 SH in the 5 position.
• substituents can be
• OH moieties in the oligomer of formula (1) may be replaced by phosphonate groups, protected by standard protecting groups, or activated to prepare
• phosphodiester analog refers to an analog of the conventional phosphodiester linkage as well as alternative linking groups. These alternative linking groups include, but are not limited to embodiments wherein the O-P(O) is replaced with P(O)S, P(O)NR 2 , P(O)R, P(O)OR', where R is H or alkyl (1-7C) and R' is alkyl (1-7C). Not all
• phosphodiester analogs in the same oligomer need to identical, the only requirement being that at least one of these linkages is a modified internucleotide linkage as described herein.
• chemotherapeutic agents An exemplary but not exhaustive list includes 4-acetylcytosine, 8-hydroxy-N 6 -methyladenine,
• gueosine 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.
• a particularly preferred analog is 5-methylcytosine
• Cme (abbreviated herein as "Cme”).
• Isosteric refers to the spatial and orientation properties of an internucleoside linkage and the fact that these properties are so similar to those of the native phosphodiester linkage that the modified oligonuceotide containing an isosteric bond will replace, substitute for, mimic and/ or hybridize with a native oligonuclotide.
• Ribose-amide refers to the internucleotide linkage that exists between two nucleobases.
• the ribose-amide internucleotide linkage has combination of ribose/ (2'-deoxy) and amino acid functionalities.
• the present invention provides novel oligonucleotide analogs containing modified amino acid/amino alcohol linkages between the bases and the backbones (phosphodiester,
• modified nucleotide linkages referred to as modified nucleotide linkages.
• the present invention is also provides novel nucleomonomers and methods for their incorporation into oligomers containing the nucleomonomers.
• the invention provides various nucleomonomer compounds having the structures of formulae 1-23.
• the oligomers of the invention are polymers comprising one or more of the subject monomer compounds joined so as to provide a structural analog of DNA or RNA.
• the oligomers of the invention comprise two or more nucleomonomers and may comprises virtually any number of nucleomonomers, although oligomers of 200 or less nucleomonomers are generally easier to synthesize.
• Compounds of formulae 1-23, may be joined to one another through 4'-5' linkages, 3'-5' linkages, and 2'-5' linkages, as can be seen in formulae 24-41.
• the nucleotide linkages in the compounds of the invention are made from amino acids serine and glycine or derivatives thereof.
• the oligonucleotides of the invention are stable in vivo, resistant to endogenous nucleases and are able to hybridize to target nucleotide sequences.
• Exemplary compounds of this invention are shown in formulae 24 through 41 and are conformationally more restricted relative to the
• This conformational restriction may, in part, contribute to the enhanced binding properties of the subject compounds to complementary polynucleotide target sequences; however, the use of the invention is not dependent upon this theory for enhanced binding properties.
• the present invention is directed to a modified oligonucleotide or derivatives thereof, wherein the furanose moiety of a natural oligonucleotide, e.g., DNA or
• RNA is replaced with amino acid/amino alcohol moiety and other modifications that comprises substitution at the amino acid positions are shown in the formulae 25 to 41.
• internucleotide linkages between adjacent nucleomonomers is a linkage between the 4' and 5' position of adjacent
• internucleotide linkage originates from 5'-position of one nucleomonomer and connects the 4'-position of adjacent monomer as exemplified by the compounds of formulae 24 - 33:
• each "R” is independently H, OH, SH, CN, CH 3 , OCH 3 , SCH 3 , ONH 2 , ONH(CH 3 ), Ph, -(CH 2 ) x -F; where "x" is 1-7 carbon and "F” is NH 2 , SH, OH, COOH, OCH 3 , SCH 3 , SPh, NOH, NOH(CH 3 ), SNH 2 , S(O)NH 2 , S(O)(O)NH 2 , CH 3 , Ph.
• each "Base” is independently a nucleoside base.
• each "R-.” is independently H, OH, SH, CN, CH 3 , OCH 3 , SCH 3 , ONH 2 , ONH(CH 3 ), Ph, -(CH 2 ) x -F; where "x" is 1-7 carbon and "F” is NH 2 , SH, OH, COOH, OCH 3 , SCH 3 , SPh, NOH, NOH(CH 3 ), SNH 2 , S(O)NH 2 , S(O)(O)NH 2 , CH 3 , Ph.
• each "R 2 " is independently H, OH, SH, CN, CH 3 , OCH 3 , SCH 3 , ONH 2 , ONH(CH 3 ), Ph, -(CH 2 ) x -F; where "x" is 1-7 carbon and "F” is NH 2 , SH, OH, COOH, OCH 3 , SCH 3 , SPh, NOH, NOH(CH 3 ), SNH 2 , S(O)NH 2 , S(O)(O)NH 2 , CH 3 , Ph.
• each "R 3 " is independently H, OH, SH, CN, CH 3 , OCH 3 , SCH 3 , ONH 2 , ONH(CH 3 ), Ph, -(CH 2 ) x -F; where "x" is 1-7 carbon and "F” is NH 2 , SH, OH, COOH, OCH 3 , SCH 3 , SPh, NOH, NOH(CH 3 ), SNH 2 , S(O)NH 2 , S(O)(O)NH 2 , CH 3 , Ph.
• each "R 4 " is independently H, OH, SH, CN, CH 3 , OCH 3 , SCH 3 , ONH 2 , ONH(CH 3 ), Ph, -(CH 2 ) x -F; where "x" is 1-7 carbon and "F” is NH 2 , SH, OH, COOH, OCH 3 , SCH 3 , SPh, NOH, NOH(CH 3 ), SNH 2 , S(O)NH 2 , S(O)(O)NH 2 , CH 3 , Ph.
• each "A” is independently (CH 2 ) x , CO, CS, S,
• each "B” is independently (CH 2 ) x , CO, CS, S,
• each "X" is independently (CH 2 ) x , CO, CS, O, S, S(O), S(O)(O), NH, NOH, NCH 3 and NR 5 , where "x" is 1- 7 carbon.
• each "Z” is independently (CH 2 ) x , CO, CS, S, S(O), S(O) (O), NH, NOH, NCH 3 and NR 5 , where "x" is 1- 7 carbon.
• R 5 is a H, OH, OMe, CN, NH, NOH, ONCH 3 , ONH 2 , ethyl, propyl, lower alkyl (1-7C), Me, heteroalkyl (1-7C), aryl(6-7C), -(CH 2 ) x F; where "x" is 1-7C, and "F” is independently H, OH, SH, OCH 3 , CN, SCH 3 , ONH 2 , ONH(CH 3 ), SNH 2 , S(O)NH 2 ,
• each "V” is independently a phosphodiester analog, phosphorothioates, methylphosphonates,
• phosphorodithioates boronphosphonates, selenophosphonates, phosphoramidates, acetamidate, oxyformamido, oxyacetamido, diisopropylsilyl, carbamate, dimethylene sulfide, dimethylene sulfoxide, dimethylene sulfone and/ or two to four atom long internucleoside linkage is selected from carbon, nitrogen, oxygen, sulfur and selenium.
• the length of the oligomer may vary from a dimer to a 200mer, or longer.
• Preferred modified internucleotide linkages include the structures for "V" are shown in Table I.
• conjugate moieties include O-cholesterol, polyethylene glycol, amino acids, intercalators, cleaving moieties (e.g., imdazole),
• crosslinking functionalities e.g., psoralen
• lipids e.g., lipids
• the conjugate moiety may independently replace one or more of R, R 1 , R 2 , R 3 , R 4 , and R 5 .
• the subject invention provides oligomer structures as indicated in formulae 34-36 and
• the linkages between adjacent nucleomonomers are 3' to 5' linkages.
• each "R” is independently H, OH, SH, CN, CH 3 , OCH 3 , SCH 3 , ONH : , ONH(CH 3 ), Ph, -(CH 2 ) x -F; where "x" is 1-7 carbon and "F” is NH 2 , SH, OH, COOH, OCH 3 , SCH 3 , SPh, NOH, NOH(CH 3 ), SNH 2 , S(O)NH 2 , S(O)(O)NH 2 , CH 3 , Ph.
• each "Base” is independently a nucleoside base.
• each "R 1 " is independently H, OH, SH, CN, CH 3 , OCH 3 , SCH 3 , ONH 2 , ONH(CH 3 ), Ph, -(CH 2 ) x -F; where "x" is 1-7 carbon and “F” is NH 2 , SH, OH, COOH, OCH 3 , SCH 3 , SPh, NOH, NOH(CH 3 ), SNH 2 , S(O)NH 2 , S(O)(O)NH 2 , CH 3 , Ph.
• each "R 2 " is independently H, OH, SH, CN, CH 3 , OCH 3 , SCH 3 , ONH 2 , ONH(CH 3 ), Ph, -(CH 2 ) x -F; where "x" is 1-7 carbon and "F” is NH 2 , SH, OH, COOH, OCH 3 , SCH 3 , SPh, NOH, NOH(CH 3 ), SNH 2 , S(O)NH 2 , S(O)(O)NH 2 , CH 3 , Ph.
• x is 1-7 carbon and "F” is NH 2 , SH, OH, COOH, OCH 3 , SCH 3 , SPh, NOH, NOH(CH 3 ), SNH 2 , S(O)NH 2 , S(O)(O)NH 2 , CH 3 , Ph.
• each "R 4" is independently H, OH, SH, CN, CH 3 ,
• each "A” is independently (CH 2 ) x , CO, CS, S,
• each "B” is independently (CH 2 ) x , CO, CS , S ,
• each "X" is independently (CH 2 ) x , CO, CS, O, S, S(O), S(O)(O), NH, NOH, NCH 3 and NR 5 .
• "x" is 1- 7.
• each "Y” is independently (CH 2 ) x , CO, CS, O, S, S(O), S(O)(O), NH, NOH, NCH 3 and NR 5 .
• "x" is 1- 7.
• each "Z” is independently (CH 2 ) x , CO, CS, S, S(O), S(O)(O), NH, NOH, NCH 3 and NR 5 .
• "x" is 1- 7.
• R 5 is a H, OH, OMe, CN, NH, NOH, ONCH 3 , ONH 2 , ethyl, propyl, lower alkyl (1-7C), Me, heteroalkyl (1-7C), aryl(6- 7C), -(CH 2 ) x F; where "x" is 1-7C, and "F” is independently H,
• each "V” is independently a phosphodiester analog, phosphorothioates, methylphosphonates,
• phosphorodithioates boronphosphonates, selenophosphonates.
• phosphoramidates and/ or two to four atom long internucleoside linkage is selected from carbon, nitrogen, oxygen, sulfur and selenium.
• the length of the oligomers may vary from a dimer to a 200mer, or longer.
• Preferred modified internucleotide linkages include the structures for "V" are shown in Table I.
• the subject invention provides oligomers having formulae 37 to 41, or variants thereof, oligomers comprising novel internucleotide linkages that are 2' ,5' linkages. These oligonucleotides are stable in vivo, have improved resistance to endogenous
• nucleases are able to hybridize to target oligonucleotide sequences.
• each "R” is independently H, OH, SH, CN, CH 3 , OCH 3 , SCH 3 , ONH 2 , ONH(CH 3 ), Ph, -(CH 2 ) x -F; where "x" is 1-7 carbon and "F” is NH 2 , SH, OH, COOH, OCH 3 , SCH 3 , SPh, NOH,NOH(CH 3 ), SNH 2 , S(O)NH 2 , S(O)(O)NH 2 , CH 3 , Ph.
• each "Base” is independently a nucleoside base.
• each "R 1 " is independently H, OH, SH, CN, CH 3 , OCH 3 , SCH 3 , ONH 2 , ONH(CH 3 ), Ph, -(CH 2 ) x -F; where "x" is 1-7 carbon and “F” is NH 2 , SH, OH, COOH, OCH 3 , SCH 3 , SPh, NOH, NOH(CH 3 ), SNH 2 , S(O)NH 2 , S(O)(O)NH 2 , CH 3 , Ph.
• each "R 2 - is independently H, OH, SH, CN, CH 3 , OCH 3 , SCH 3 , ONH 2 , ONH(CH 3 ), Ph, -(CH 2 ) x -F; where "x" is 1-7 carbon and "F” is NH 2 , SH, OH, COOH, OCH 3 , SCH 3 , SPh, NOH, NOH(CH 3 ), SNH 2 , S(O)NH 2 , S(O)(O)NH 2 , CH 3 , Ph.
• each "R 3 . is independently H, OH, SH, CN, CH 3 , OCH 3 , SCH 3 , ONH 2 , ONH(CH 3 ), Ph, -(CH,) x -F; where "x" is 1-7 carbon and "F” is NH 2 , SH, OH, COOH, OCH 3 , SCH 3 , SPh, NOH, NOH(CH 3 ), SNH 2 , S(O)NH 2 , S(O)(O)NH 2 , CH 3 , Ph.
• each "R 4 . is independently H, OH, SH, CN, CH 3 , OCH 3 , SCH 3 , ONH 2 , ONH(CH 3 ), Ph, -(CH 2 ) x -F; where "x" is 1-7 carbon and "F” is NH 2 , SH, OH, COOH, OCH 3 , SCH 3 , SPh, NOH, NOH(CH 3 ), SNH 2 , S(O)NH 2 , S(O)(O)NH 2 , CH 3 , Ph.
• each "A” is independently (CH 2 ) x , CO, CS, S,
• each "B” is independently (CH 2 ) x , CO, CS, S, S(O), S(O)(O), NH, NOH, NCH 3 , NR 5 and Se; where "x" is 1- 7 carbon.
• each "X” is independently (CH 2 ) x , CO, CS , O, S, S(O), S(O)(O), NH, NOH, NCH 3 and NR 5 ; where "x" is 1- 7 carbon.
• each "Z” is independently (CH 2 ) x , CO, CS, S,
• R b is a H, OH, OMe, CN, NH, NOH, ONCK 3 , ONH,, ethyl, propyl, lower alkyl (1-7C), Me, heteroalkyl (1-7C), aryl(67C), -(CH 2 ) x F; where "x" is 1-7C, and "F” is independently H, OH, SH, OCH 3 , CN, SCH 3 , ONH 2 , ONH(CH 3 ), SNH 2 , S(O)NH 2 ,
• each "V” is independently a phosphodiester analog, phosphorothioates, methylphosphonates,
• phosphorodithioates boronphosphonates, selenophosphonates, phosphoramidates and/ or two to four atom long internucleoside linkage is selected from carbon, nitrogen, oxygen, sulfur and selenium.
• the length of the oligomer varies from dimer to 200mer.
• Preferred modified internucleotide linkages include the structures for "V" are shown in Table I.
• the subject invention is directed to an oligomer of the following formulae (formula 42) and monomeric constituents thereof
• Y is selected from the group consisting of CH 2 , CO, COOH, CS, and SO 2 ,
• Y' is selected from the group consisting of CH 2 , CO, COOH, CS, and SO 2 ,
• Z is selected from the group consisting of O, S, NH, and CH 2
• R is selected from the group consisting ofCH 2 OH, CH 2 NH 2 , CH 2 NHCHO, CONH 2 , and COOH,
• B is a nucleoside base
• Y is selected from the group consisting of CH 2 , CO, COOH, CS, and SO 2 ,
• Y' is selected from the group consisting of CH 2 , CO, COOH, CS , and SO 2 ,
• Z is selected from the group consisting of O, S, NH, and CH 2 ,
• R is selected from the group consisting ofCH 2 OH, CH 2 NH 2 ,
• the invention provides methods for treating diseases mediated by the presence of a nucleotide sequence which comprise administering to a subject in need of such treatment an amount of the above modified
• nucleotide sequence effective to inactivate the nucleotide sequence.
• oligonucleotides of the invention at least one of the phosphodiester groups included within the "Vs" of Formulae 24-41 is substituted by the modified internucleoside linkages described herein. Desirably, multiple phosphodiester linkages in the unmodified oligonucleotide are substituted by the modified internucleoside linkage may be used repeatedly in this structure, or, if desired, a variety of modified
• internucleotide linkages may be used in an individual
• oligonucleotide In a preferred embodiment of the subject oligonucleotides these substituent linkages are non-chiral so as to enhance the ability of the oligonucleotide to hybridize to a desired target; however, useful compounds of the
• Perferred modified internucleotide linkages include the structures for "V" are shown in the Table 1.
• R 3 is a H, OH, OMe, CN, NH, NOH, ONCH 3 , ONH 2 , ethyl, propyl, lower alkyl (1-7C), Me, heteroalkyl (1-7C), aryl(6-7C), -(CH 2 ) x F; where "x" is 1-7C, and "F” is independently H, OH, SH, OCH 3 , CN, SCH 3 , ONH 2 , ONH(CH 3 ), SNH 2 , S(O)NH 2 ,
• conjugate one or more moieties may be joined to the linkage so as to produce an oligomer conjugate.
• Suitable conjugate moieties include, O-cholesterol, polyethylene glycol, amino acids,
• intercalulators cleaving moieties (e.g., imdazole),
• crosslinking functionalities e.g., psoralen
• lipids e.g., lipids, peptides, alkylating agents, hydroxamates, and fluorescent labels.
• 4'-5' linkages include
• oligomers of the invention are not limited to
• oligomers of homogeneous linkage type and that alternating or randomly distributed substitute linkages including the 2', 5' linkages are included. Since the oligomers of the invention can be synthesized one nucleomonomer residue at a time, each individual linkage, and/or substitute linkage, and the nature of each individual "Base" substituent may be selected
• oligonucleotides having a desired sequence independently so as to produce oligonucleotides having a desired sequence.
• the oligomers of the invention may contain any desired number of the substitute linkages. These substitute linkages may be identical to each other or different by virtue of the embodiments chosen for "V" including other noninvention substitute linkages. Since the oligomers are prepared
• the substitute linkages of the invention alternate in a regular pattern.
• one substitute linkage is followed by two
• phosphodiester linkages followed by one invention substitute linkage etc. Additional embodiments include, for example, alternating linkages such as a substitute linkage followed by a phosphodiester analog (e.g., thioate, etc.), followed by a substitute linkage of the invention followed by a phosphodiester analog (e.g., thioate, etc.), followed by a substitute linkage of the invention followed by a phosphodiester analog (e.g., thioate, etc.), followed by a substitute linkage of the invention followed by a
• Oligomers of the invention may comprise a one-by-one alternation of the two types of substitute linkages.
• Oligomers of the invention comprising more than one type of linkage may have any of a number of regular patterns formed by alternations between the different linkage types present between the subunits of the oligomer.
• oligonucleotide analog For example, in standard DNA (or RNA) the sequences are generally denoted by the sequence of bases alone, such as, for example, ATG CGC TGA. In general, it is simply stated in advance whether this represents an RNA or DNA sequence. A corresponding notation system is used herein so as to represent oligonucleotide analogs with a given base sequence.
• Oligomers of the invention may also comprise of various modifications in addition to the substitute linkages of the invention. Additional modifications include oligomers where (i) one or more nucleomonomer residues are modified at the 2', 3', 4', and 5' positions, (ii) one or more covalent
• crosslinking moieties are incorporated, (iii) other
• noninvention substitute linkages are included, (iv) other base analogs, such as 8-oxo-N 6 -methyladenine, are included and (v) conjugates such as intercalating agents or polylysine that respectively enhance binding affinity to target nucleic acid sequences or that enhance association of the oligomer with cells are included.
• sequence-specific polynucleotide binding properties of the oligomers of the invention for single-stranded and duplex targets is compatible with further modifications to the oligomer. These further modifications may also confer other useful properties such as stability to nuclease cleavage (e.g. in a domain of an oligomer of the invention having
• the oligomers of the invention may comprise one or more substitute linkages such as sulfide or sulfone linkages
• oligomers having (1) at least one substitute linkage and an amino acid that is linked to an adjacent monomer and (2) one or more non-invention substitute linkages selected from the group consisting of phospnorotnioate,
• exemplary oligomers would include (1) an oligomer having invention substitute linkages at the 3' and/or 5' ends and phosphorothioate
• pyrimidine bases e.g. adenine, guanine, cytosme, thymine, or uracil
• oligomers having invention substitute linkages and one or more bases that enhance binding affinity
• oligomer e.g. 5-methylcytosme, 5'(1-propynyl) uracil, 5-(1-propynl) cytosme.
• oligomers containing nucleomonomer residues linked via hydroxamates are also included.
• oligomers of the invention may be formed using nucleomonomers of the invention alone or in combination with conventional nucleomonomers and synthesized using standard solid phase (or solution phase) oligomer synthesis techniques, which are now commercially available.
• solid phase (or solution phase) oligomer synthesis techniques which are now commercially available.
• oligomers may be synthesized by a method comprising the steps of: synthesizing a nucleomonomer or oligomer synthon having a protecting group and a base and a coupling group capable of coupling to a nucleomonomer or oligomer;
• the oligomers of the present invention may be of any length including those of greater than 40, 50, 100, 200 or 500 nucleomonomers. In general, preferred oligomers contain 2-30 nucleomonomers. Lengths of greater than or equal to about 8 to 20 nucleomonomers may be useful for therapeutic or
• nucleomonomers are Specifically included in the present specification.
• Oligomers having a randomized sequence and containing about 6, 7 or 8 nucleomonomers may be used as primers that are used in cloning or amplification protocols that use random sequence primers, provided that the oligomer contains about 1 or 2 residues at the 3' end that can serve as a primer for polymerases or reverse transcriptases or that otherwise do not interfere with polymerase activity.
• the oligomers of the invention may comprise
• substitute linkages include, but are not limited to,
• linkages are well known. Particularly preferred substitute linkages for use in the oligomers of the present invention include phosphodiester, phosphorothioate, methylphosphonate and thionomethylphosphonate substitute linkages.
• Phosphorothioate and methylphosphonate substitute linkages confer added stability to the oligomer need be identical, particularly preferred oligomers of the invention contain one or more phosphorothioate or methylphosphonate substitute linkages.
• Oligomers of the invention and the segments thereof may be synthesized using methods that are known to the personof ordianry skill in the art.
• the synthetic methods known in the area and described herein can be used to synthesize oligomers containing substitute linkages of the invention, as well as other linkages or substitute linkages known in the art, using appropriately protected nucleomonomers.
• Methods for the synthesis of oligomers having phosphorous containing linkages are found, for example, in Froehler, B., et al., Nuclei c Acids Res . , 1986, 11, 5399-5467; Nuclei c Acids Res . , 1988, 16, 4831-4839; Nucleosides & Nucleotides, 1987, 6, 287-291;
• Oligomers containing linkages of the present invention are also conveniently synthesized by preparation of dimer or trimer compounds by solution phase chemistry followed by conversion of the synthon to a derivative that is incorporated into oligomers by either solid or solution phase chemistry.
• Typical synthons are 5' DMT or MMT blocked 3' phosphonate or phosphoramidate derivatives which are prepared by standard methods (see: Gait, M.J. ed., Oligonucleotide Synthesis; A
• Synthons that are included in the scope of the present invention include dimers, trimers, tetramers, hexamers and longer oligomer made by solid or solution phase synthesis.
• Trimers and longer synthons may contain more than one type of linkage.
• the synthons may include any base as described above or 2', 3', 4' and 5' groups such as OH, DMTO, MMTO, O-allyl, phosphate, a phosphonate or an amidite as described above.
• Ribose-amide oligonucleotides could be synthesized by using standard solid phase peptide synthesis (Fmoc chemistry) conditions (see figure 26). Blocking Groups For the Synthesis of the Compound of the
• Suitable coupling groups are, for example, H-phosphonate, a methylphosphonomidite, or a phosphoramidite.
• Phosphoramidites that can be used include ⁇ - cyanoethylphosphoramidites (preferred).
• Methylphosphonamidites alkylphosphonamidites (including ethylphosphonamidites and propylphosphonamidites) can also be used.
• Exemplary phosphoramidites are shown in figures 1 to 21.
• Suitable "coupling groups" at the 2 ' , 3 ' , 4 ' or 5' position for oligomer synthesis via phosphoramidite triester chemistry include N,N-diisopropylamino- ⁇ -cyanoethoxyphosphine, N- N,diisopropylamino-methoxyphosphine, N, N-diethylamino- cyanoethoxyphosphine, and (N-morpholino)-methoxyphosphine
• coupling groups such as N,N-diisopropylamino-methyl-phosphine or N,N-diethylamino-methyl-phosphine can also be used to prepare methylphosphonates.
• Methylphosphonate oligomers can be conveniently synthesized using coupling groups such as N,N-diisopropylamino-methylphosphoramidite.
• nucleomonomer amidites of the invention can be accomplished by conventional methods (for example, Gryaznov, S.M., et al, Nucl Acids Res . , 1992, 20 , 1879-1882; Vinayak, R., et al, Nucl
• Protecting groups such as diisobutylformamidine, benzoyl, isobutyryl, FMOC, dialkylformamidine, dialkylacetamidine or other groups known in the art can be used to protect the exocyclic nitrogen of the cytosine, adenine or guanine
• cytidine can be directly
• Suitable protecting groups are DMT (dimethoxy trityl), Bz (benzoyl), Bu (isobutyryl), phenoxyacetyl, MMT
• TBS t-butyldimethylsilyl
• TBDPS t-butyldiphenylsilyl
• Preferred protecting groups are Bz (benzoyl), DMT
• nucleomonomers and oligomers of the invention can be derivatized to such
• blocking groups as indicated in the relevant formulas by methods known in the art.
• the subject invention also provides for "conjugates" of the oligomers of the invention.
• Conjugates of conventional oligomers are known to the person of ordinary skill in the art.
• the oligomers of the invention may be covalently linked to various moieties such as, for example, intercalators, and compounds which interact specifically with the minor groove of the DNA double helix.
• moieties for conjugation to the subject oligomers include, labels, (e.g., radioactive, fluorescent, enzyme) or moieties which facilitate cell association using cleavable linkers and the like.
• Suitable radiolabels include 32 P, 35 S, 3 H, 131 I and 14 C ; and suitable fluorescent labels include fluorescence, resorufin, rhodamine, BODIPY (Molecular Probes) and Texas red; suitable enzymes include alkaline phosphatase and horseradish
• fragments, asialoglycoprotein, transferrin and the HIV Tat protein can also conveniently be linked to the oligomers of the invention.
• intercalators such as acridine or psoralen can be linked to the oligomers of the invention through any available - OH or -SH, e.g., at the terminal 5'-position of the oligomer, the 2' -positions of RNA, or an OH, NH 2 , COOH or SH incorporated into the 5-position of pyrimidines .
• a derivatized form which contains, for example,
• Conjugates including polylysine or lysine can be synthesized as described and can further enhance the
• substituents can be attached, including those bound through linkages or substitute linkages.
• the -OH moieties in the oligomers can be replaced by phosphate groups, protected by standard protecting groups, or coupling groups to prepare additional linkages to other nucleomonomers, or can be bound to the conjugated substituent.
• the 5'-terminal OH can be phosphorylated; the 2'-OH or OH substituents at the 3'- terminus can also be phosphorylated.
• the hydroxyls can also be derivatized to standard protecting groups.
• Oligomers of the invention can be covalently derivatized to moieties that facilitate cell association using cleavable linkers.
• Suitable conjugates also include solid supports for oligomer synthesis and to facilitate detection of nucleic acid sequences. Solid supports include, but are not limited to, silica gel, controlled pore glass, polystyrene, and magnetic glass beads.
• Derivatives can be made by substitution on the sugars.
• inventions are the 2'-O-allyl or 3'-allyl group appears to enhance permeation ability and stability to nuclease
• the oligomers of the invention may also contain one or more "substitute linkages", in addition to the 2'-5' , 3'-5' and 4'-5' linkages disclosed herein, which are generally understood in the art.
• substitute linkages include phosphorothioate, methylphosphonate, thionomethylphosphonate, phosphorodithioate, alkylphosphonates, morpholino sulfamide, boranophosphate (-O-P(OCH 3 )(BH 3 )-O-), siloxane (-O-Si(X 4 ) (X 4 )- O-; X 4 is 1 - 6C alkyl or phenyl) and phosphoramidate
• Substitute linkages that can be used in the oligomers disclosed herein also include the sulfonamide (-O-SO 2 -NH-), sulfide (-CH,-S-CH 2 -), sulfonate (-O- SO,-CH 2 -). carbamate (O-C(O)-NH-, -NH-C(O)-O-),
• substitute linkages such as a formacetal linkage, -O- CH 2 -O-
• substitute linkages are linked to either the 4', 3', 2' carbon of a nucleomonomer on the left side and to the 5' carbon of a nucleomonomer on the right side.
• the designations of a 4', 3', 2' or 5' carbon can be modified accordingly when a structure other than ribose, deoxyribose or arabinose is linked to an adjacent
• nucleomonomer Such structures include xylose, a hexose, morpholino ring, carbocyclic ring (e.g. cyclopentane) and the like.
• Substitute linkage(s) can be utilized in the oligomers for a number of purposes such as to rurther facilitate binding with complementary target nucleic acid sequences and/or to increase the stability of the
• compounds of the invnetion include not only the naturally occurring purine and pyrimidine bases, but also analogs of these heterocyclic bases and tautomers thereof.
• Such analogs include alkylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles.
• Such “analogous purines” and “analogous pyrimidines” or purine or pyrimidine analogs are those generally known in the art, some of which are used as chemotherapeutic agents.
• An exemplary, but not exhaustive, list includes N 4 N 4 - ethanocytosine, 7-deazaxanthosine, 7- deazaguanosine, 8-oxo-N 6 -methyladenine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,
• 5-carboxymethylaminomethyl-2-thiouracil 5- carboxymethylaminomethyl uracil, inosine, N 6 -isopentenyladenine, 1-methyladenine, 2-methylguanine, 5-methylcytosine,
• pyrimidine analogs including 6-azacytosine, 6-azathymidi.ne and
• Preferred bases include adenine, guanine, thymine, uracil, cytosine, 5-methylcytosine, 5-(1-propynyl) uracil, cytosine, 5-methylcytosine, 5-(1-propynyl) uracil, 5-(1-propynyl) cytosine, 8-oxo-N 6 -methyladenine, 7-deaza-7-methylguanine, 7-deaza-7-methyladenine and 7-deazaxanthosine.
• oligomers of the invention is a moiety which is capable of effecting at least one covalent bond between the oligomer and the duplex. Multiple covalent bonds can also be formed by providing a multiplicity of such crosslinking moieties.
• the covalent bond is preferably to a base residue in the target strand, but can also be made with other portions of the target, including the saccharide or phosphodiester.
• the reaction nature of the moiety which effects crosslinking determines the nature of the target in the duplex.
• Preferred crosslinking moieties include acylating and alkylating agents, and, in particular, those positioned relative to the sequence specificity-conferring portion so as to permit reaction with the target location in the strand.
• heterocycle need not be a purine or pyrimidine; indeed the pseudo-base to which the reactive function is attached need not be a heterocycle at all. Any means of attaching the reactive group is satisfactory so long as the positioning is correct.
• the symbol 3' 5' indicates a stretch of oligomer in which the linkages are consistently formed between the 5'-hydroxyl of the amino acid residue of the nucleomonomer to the left with the 3'- (or 2'- for
• 5' 3' indicates a stretch of oligomer in the opposite orientation wherein the linkages are formed between the 3'-hydroxyl of the amino acid residue of the left nucleomonomer and the 5'-hydroxyl of the amino acid residue of the nucleomonomer on the right, thus leaving the
• the invention also provides for various salts of all compounds disclosed herein, including pharmaceutically acceptable salts for administration to an animal or human.
• Pharmaceutically acceptable salts and such salt forming materials are well known in the art.
• Pharmaceutically acceptable salts are preferably metal or ammonium salts of the oligomers of the invention and include alkali or alkaline earth metal salts, e.g., the sodium, potassium, magnesium or calcium salt; or advantageously easily crystallizing ammonium salts derived from ammonia or organic amines, such as mono-, di- or tri-lower (alkyl, cycloalkyl or hydroxyalkyl)-amides, lower alkylenediamines or lower (hydroxyalkyl or arylalkyl)alkylammonium bases, e.g. methylamine, diethylamine,
• oligomers of the invention may form acid addition salts, preferably of therapeutically acceptable inorganic or organic acids, such as strong mineral acids, for example hydrophilic, e.g., hydrochloric or
• hydrobromic acid sulfuric, phosphoric; aliphatic or aromatic carboxylic or sulfonic acids, e.g., formic, acetic, propionic, succinic, glycollic, lactic, malic, tartaric, gluconic, citric, ascorbic, maleic, fumaric, hydroxymaleic, pyruvic, phenylacetic, benzoic, 4-aminobenzoic, anthranilic, 4-hydroxynbenzoic, salicylic, 4-aminosalicylic, methanesulfonic, ethanesulfonic, hydroxyethanesulfonic, benzenesulfonic, sulfanilic or cyclohexylsulfamic acid and the like.
• oligomers of the invention are capable of significant single-stranded or double-stranded target nucleic acid binding activity to form duplexes, triplexes or other forms of stable association, with naturally occurring
• the oligomers of the invention may be used in most procedures that employ conventional oligomers.
• the oligomers of the invention may be used as, for example, polynucleotide hybridization probes, primers for the polymerase chain reaction and similar cyclic amplification reactions, sequencing primers, and the like.
• the oligomers of the invention may also be used in the diagnosis and therapy of diseases.
• Therapeutic applications of the oligomers of the invention include the specific inhibition of the expression of genes (or inhibit translation of RNA sequences encoded by those genes) that are associated with either the establishment or the maintenance of a
• the oligomers of the invention may be used to mediate
• RNAs encoded by those genes that can be targeted through antisense employing the oligomers include those that encode enzymes, hormones, serum proteins, transmembrane proteins, adhesion molecules (LFA-1, GPII b /III a , ELAM-1, VACM- 1, ICAM-1, E-selection, and the like), receptor molecules including cytokine receptors, cytokines (IL-1, IL-2, IL-3, IL- 4, IL-6 and the like), oncogenes, growth factors, and
• Target genes or RNAs can be associated with any pathological condition such as those associated with
• Oligomers of the present invention are suitable for use in both in vivo and ex vivo therapeutic applications.
• Indications for ex vivo uses include treatment of cells such as bone marrow or peripheral blood in conditions such as leukemia (chronic myelogenous leukemia, acute lymphocytic leukemia) or viral infection.
• Target genes or RNAs encoded by those genes that can serve as targets for cancer treatments include oncogens, such as ras, k-ras, bcl-2, c-myb, bcr, c- myc, c-abl or overexpressed sequences such as mdm2, oncostatin M, IL-6 (Kaposi's sarcoma), HER-2 and translocations such as bcr-abl.
• oncogens such as ras, k-ras, bcl-2, c-myb, bcr, c- myc, c-abl or overexpressed sequences such as mdm2, oncostatin M, IL-6 (Kaposi's sarcoma), HER-2 and translocations such as bcr-abl.
• Viral gene sequences or RNAs encoded by those genes such as polymerase or reverse transcriptase genes of
• herpesviruses such as CMV, HSV-1, HSV-2, retroviruses such as
• HTLV-1 HIV-1
• HIV-2 or other DNA or RNA viruses
• HBV HBV
• HPV HPV
• VZV influenza virus
• adenoviruses flaviviruses
• oligomers of the invention include (1) modulation of inflammatory responses by modulating expression of genes such as IL-1 receptor, IL-1, ICAM-1 or E-Selection that play a role in mediating inflammation and (2) modulation of cellular proliferation in conditions such as arterial occlusion (restenosis) after angioplasty by modulating the expression of (a) growth or mitogenic factors such as nonmuscle myosin, myc, fox, PCNA, PDGF or FGF or their receptors, or (b) cell proliferation factors such as c-myb.
• genes such as IL-1 receptor, IL-1, ICAM-1 or E-Selection that play a role in mediating inflammation
• modulation of cellular proliferation in conditions such as arterial occlusion (restenosis) after angioplasty by modulating the expression of (a) growth or mitogenic factors such as nonmuscle myosin, myc, fox, PCNA, PDGF or FGF or their receptors, or (b) cell proliferation factors such as c
• suitable proliferation factors or signal transduction factors such as TGFx, IL-6, gINF, protein kinase C, tyrosine kinases
• EGF receptor EGF receptor
• TGFa or MHC alleles may be targeted in autoimmune diseases. Delivery of oligomers of the invention into cells can be enhanced by any suitable method including calcium phosphate, DMSO, glycerol or dextran transfection, electroporation or by the use of cationic anionic and/or neutral lipid compositions or liposomes by methods described (International Publications
• the oligomers can be introduced into cells by complexion with cationic lipids such as DOTMA (which may or may not form liposomes) which complex is then contacted with the cells.
• cationic lipids include but are not limited to N- (2, 3-di(9-(Z)-octadecenyloxyl))-prop-1-yl-N,N,N- trimethylammonium (DOTMA) and its salts, 1-O-oleyl-2-O-oleyl- 3-dimethylaminopropyl- ⁇ -hydroxyethylammonium and its salts and
• Enhanced delivery of the invention oligomers can also be mediated by the use of (i) viruses such as Sendai virus .
• anionic, neutral or pH sensitive lipids using compounds including anionic phospholipids such as phosphatidyl glycerol, cardiolipin, phosphatidic acid or
• transfection refers to any method that is suitable for
• oligomers into cells.
• Any reagent such as a lipid or any agent such as a virus that can be used in transfection protocols is collectively referred to herein as a "permeation enhancing agent”.
• Delivery of the oligomers into cells can be via cotransfection with other nucleic acids such as (i) expressable DNA fragments encoding a protein(s) or a protein fragment or (ii) translatable RNAs that encode a protein (s) or a protein fragment.
• oligomers of the invention can thus be incorporated into any suitable formulation that enhances delivery of the oligomers into cells.
• suitable pharmaceutical formulations also include those commonly used in applications where
• Compounds are delivered into cells or tissues by topical administration.
• Compounds such as polyethylene glycol, propylene glycol, azone, nonoxonyl-9, oleic acid, DMSO, polyamines or lipopolyamines can be used in topical
• the invention oligomers can be conveniently used as reagents for research or production purposes where inhibition of gene expression is desired. There are currently very few reagents available that efficiently and specifically inhibit the expression of a target gene by any mechanism. Oligomers that have been previously reported to inhibit target gene expression frequently have nonspecific effects and; or do not reduce target gene expression to very low levels (less than about 40% of uninhibited levels).
• the oligomers as described herein constitute a reagent that may be used in methods of inhibiting expression of a selected protein or proteins in a subject or in cells wherein the proteins are encoded by DNA sequences and the proteins are translated from RNA sequences, comprising the steps of: introducing an oligomer of the invention into the cells; and permitting the oligomer to form a triplex with the
• the methods and compound of the present invention are suitable for modulating gene expression in both procaryotic and eucaryotic cells such as bacterial, fungal parasite, yeast and mammalian cells.
• RNase H "competent” or RNase H "incompetent” oligomers can be easily designed using the substitute linkages of the invention.
• RNase H competent oligomers can comprise one or more RNase H competent domains comprised of linked RNase H competent nucleomonomers. Oligomers having modifications such as 2'-substitutions (2'-O-allyl and the like) or certain uncharged linkages (methylphosphonate, phosphoramidate and the like) are usually incompetent as a substrate that is
• RNase H competence can facilitate antisense oligomer function by degrading the target RNA in an RNA-oligomer duplex (Dagle, J.M. et al, Nucl
• the enzyme cleaves RNA in RNA-DNA duplexes.
• an oligomer In order to retain RNase H competence, an oligomer requires a RNase H competent domain of three or more competent contiguous nucleomonomers located within it (Quartin, R.S., et al, Nucl Acids Res., 1989, 17, 7253-7262). Design of
• oligomers resistant to nuclease digestion will have terminal linkage, sugar and/or base modifications to effect nuclease resistance.
• the oligomers can be designed to have modified nucleomonomer residues at either or both the 5'- and/or 3'- ends, while having an internal RNase H competent domain.
• Exemplary oligomers that retain RNase H competence would generally have uniform polarity and would comprise about 2 to about 12 nucleomonomers at the 5 ' - end and at the 3'- end which stabilize the oligomer to nuclease degradation and about three to about 26 nucleomonomers that function as a RNase H competent domain between the RNase H incompetent 3' and 5'- ends. Variations on such an oligomer would include (1) a shorter RNase H competent domain comprising 1 or 2 RNase H competent linkages or substitute linkages, (2) a longer RNase H incompetent domain comprising up to 15, 20 or more
• Oligomers containing as few as about 8 nucleomonomers may be used to effect inhibition of target protein (s) expression by formation of duplex or triplex structures with target nucleic acid sequences.
• linear oligomers used to inhibit target protein expression via duplex or triplex may be used to effect inhibition of target protein (s) expression by formation of duplex or triplex structures with target nucleic acid sequences.
• Oligomers containing substitute linkages of the invention can be conveniently circularized as described (International Publication No. WO 92/19732; Kool, E.T. J Am Chem Soc., 1991, 113, 6265-6266; Prakash, G. et al, J Am Chem Soc , 1992, 114, 3523-3527). Such oligomers are suitable for binding to
• Circular oligomers can be of various sizes. Such oligomers in a size range of about 22-50 nucleomonomers can be conveniently prepared.
• the circular oligomers can have from about three to about six nucleomonomer residues in the loop region that separate binding domains of the oligomer as described
• Oligomers can be enzymatically
• the oligomers can be utilized to modulate target gene expression by inhibiting the interaction of nucleic acid binding proteins responsible for modulating transcription
• the oligomers are thus suitable as sequence- specific agents that compete with nucleic acid binding
• proteins including ribosomes, RNA polymerases, DNA
• oligomers can thus be used to increase target protein synthesis through mechanisms such as binding to or near a regulatory site that transcription factors use to repress expression or by inhibiting the expression of a
• modifications that enhance binding affinity can be designed to contain secondary or tertiary structures, such as pseudoknots or pseudo-half-knots (Ecker, D.J. et al, Science, 1992, 257, 958-961). Such structures can have a more stable secondary or tertiary structure than corresponding unmodified oligomers.
• Such structures can be used to mimic structures such as the
• HIV TAR structure in order to interfere with binding by the
• HIV Tat protein (a protein that binds to TAR).
• a similar approach can be utilized with other transcription or
• the invention oligomers can be used to (1) disrupt or (2) bind to such structures as a method to (1) interfere with or (2) enhance the binding of proteins to nucleic acid structures.
• the oligomers of the invention can also be applied as therapeutic or diagnostic agents that function by direct displacement of one strand in a duplex nucleic acid.
• chromosomal DNA or duplex viral DNA, RNA or hybrid DNA/RNA is possible for oligomers with a high binding affinity for their complementary sequence is not great enough to efficiently displace a DNA or RNA strand in a duplex.
• Types of target nucleic acids include but are not limited to (i) gene
• sequences including exons, introns, exon/intron junctions, promoter/enhancer regions and 5' or 3' untranslated regions,
• regions of nucleic acids that utilize secondary structure in order too function e.g. the HIV TAR stem-loop element or tRNAs
• nucleic acids that serve structural or other functions such as telomeres, centromeres or replication
• oligomers can be synthesized with discrete functional domains wherein one region of an oligomer binds to a target by D-looping while an adjacent region binds a target molecule by say, forming a triple helix or binding as an aptamer to a protein.
• a D- looping oligomer can bind to each strand in a duplex by switching the strand to which the oligomer binds (i.e. by having one region of the oligomer that binds to one strand and another region that binds to the complementary strand).
• the controlling elements that dictate the mode of binding are the sequence of the oligomer and the inherent affinity built into the oligomer. Base
• D-loop structures are formed in nature by enzyme-mediated processes (Harris, L.D. et al., et al., J Biol Chem . , 1987, 262, 9285- 9292) or are associated with regions where DNA replication occurs (Jacobs, H.T. et al., Nucl Acids Res, 1989, 17, 8949- 8966). D-loops that arise from the binding of oligomers can result from a one or two step process. Direct displacement of a target strand will give rise to a D-loop by a single binding event. However, D-looping can also occur by forming a triple helix which facilitates a strand displacement envent leading to the D-loop.
• Ribozymes containing substitute linkages of the invention can be designed in order to design species with altered
• Ribozymes that cleave single stranded RNA or DNA have been described.
• Therapeutic applications for ribozymes have been Dostulated (Sarver, M. et al., Science, 1990, 247,
• ribozymes having nuclease stable targeting domains containing substitute linkages of the invention can have higher affinity, while maintaining base pairing specificity, for target sequences. Because of the higher affinity and/or nuclease stability of the invention substitute linkages shorter recognition domains in a ribozyme (an advantage in manufacturing) can be designed which can lead to more favorable substrate turnover (an advantage in ribozyme function).
• the oligomers can be formulated for a variety of modes of administration, including systemic, topical or localized administration.
• the oligomer active ingredient is generally combined with a carrier such as a diluent or
• excipient which can include fillers, extenders, binders, wetting agents, disintegrants, surface-active agents, or lubricants, depending on the nature of the mode of
• Typical dosage forms include tablets, powders, liquid preparations including suspensions, emulsions and solutions, granules, capsules and suppositories, as well as liquid preparations for injections, including liposome preparations.
• the oligomers of the invention are formulated in liquid solutions, preferably in
• oligomers can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included. Dosages that can be used for systemic
• administration preferably range from about 0.01 mg/Kg to 50 mg/Kg administered once or twice per day.
• different dosing schedules can be utilized depending on (i) the potency of an individual oligomer at inhibiting the activity of its target DNA or RNA, (ii) the severity or extent of a
• Systemic administration can also be by transmucosal or transdermal means, or the compounds can be administered orally.
• transmucosal or transdermal administration penetrates appropriate to the barrier to be permeated are used in the formulation. Such penetrates are generally known in the art, and include, for example, bile salts and fusidic acid derivatives for transmucosal administration.
• detergents can be used to facilitate permeation.
• Transmucosal administration can be through use of nasal sprays, for
• the oligomers are formulated into conventional oral administration forms such as capsules, tablets, and tonics.
• the oligomers of the invention For topical administration, the oligomers of the
• oligomers for ocular indications such as viral infections would be based on standard compositions known in the art.
• the oligomers of the invention can be used as diagnostic reagents to detect the presence or absence of the target nucleic acid sequences to which they specifically bind.
• the enhanced binding affinity of the invention oligomers is an advantage for their use as primers and probes. Diagnostic tests can be conducted by hybridization through either double or triple helix formation which is then detected by conventional means.
• the oligomers can be labeled using radioactive, fluorescent, or chromogenic labels and the presence of label bound to solid support detected.
• the presence of a double or triple helix can be detected by antibodies which specifically recognize these forms.
• Means for conducting assays using such oligomers as probes are generally known.
• the use of oligomers of the invention substitute linkages as diagnostic agents by triple helix formation is advantageous since triple helices form under mild conditions and the assays can thus be carried out without subjecting test specimens too harsh conditions. Diagnostic assays based on detection of RNA for identification of bacteria, fungi or protozoa sequences often required isolation of RNA from samples or organisms grown in the laboratory, which is laborious and time
• the oligomer probes can also incorporate additional modifications such as modified sugars and/or substitute
• the invention probes can also contain linkers that permit specific binding to alternate DNA strands by
• Oligomers of the invention are suitable for use in
• diagnostic assays that employ methods wherein either the oligomer or nucleic acid to be detected are covalently
• the oligomers are also suitable for use in
• Oligomers of the invention containing a 3' terminus that can serve as a primer are compatible with polymerases used in polymerase chain reaction methods such as the Taq or Vent TM
• Oligomers of the invention can thus be utilized as primers in PCR protocols.
• the oligomers of the invention are useful as primers that are discrete sequences or as primers with a random sequence.
• Random sequence primers can be generally about 6, 7, or 8 nucleomonomers in length. Such primers can be used in various nucleic acid amplification protocols (PCR, ligase chain reaction, etc.) or in cloning protocols.
• the substitute linkages of the invention generally do not interfere with the capacity of the oligomer to function as a primer. Oligomers of the invention having 2'-modifications at sites other than the 3' terminal residue, other modifications that render the oligomer RNase H incompetent or otherwise nuclease stable can be advantageously used as probes or primers for RNA or DNA sequences in cellular extracts or other solutions that contain nucleases.
• the oligomers can be used in protocols for amplifying nucleic acid in a sample by mixing the oligomer with a sample containing target nucleic acid, followed by hybridization of the oligomer with the target nucleic acid and amplifying the target nucleic acid by PCR, LCR or other
• oligomers derivatized to chelating agents such as
• EDTA, DTPA or analogs of 1,2-diaminocyclohexane acetic acid can be utilized in various invitro diagnostic assays as
• oligomers of the invention can be derivatized with crosslinking agents such as 5-(3-iodoacetamidoprop-1-yl) 2'-deoxyuridine or 5-(3-(4-bromobutyramido) prop-1-yl)-2'-deoxyuridine and used in
• oligomers of the invention can be synthesized using reactions known in the art of oligonucleotide derivative synthesis. See e.g. Flandor, J. and Yam, S.Y., Tetrahedron
• the substitute linkages of the invention can vary so as to contain one or more nitrogen, sulfur, and/or oxygen atoms in their structure.
• the positions of these atoms in the substitute linkage can vary from the "5'" end, to the "middle” to the "2"' or “3"' and "4"' end.
• the first five steps shown in Figure 1 relate to the preparation of isobutryl protected serinol amino acid alcohol.
• step 1 of Figure 1 the amino group of the serine amino acid is protected by reacting 1 with di-tert-butyl dicarbonate to yield compound 2. Other equivalent protecting groups may be used.
• step 2 the ⁇ -hydroxyl group of Compound 2 is blocked with dihydropyran to give fully
• Thymine acetic acid 7 was prepared as described in the literature (see: L. Kosynkina, W. Wang and T. C.
• thymine acetaldehyde 13 was produced by the treatment of thymine with bromoacetaldehyde dimethylactal followed by hydrolysis of 12 with aqueous TFA.
• the starting material is a ⁇ - substituted amino acid 18.
• the substituted amino acid could be transformed into the phosphoroamidite building block 27 by following the procedure of the steps used in Figures 1 and 2.
• the bisamine 48 could then be converted to a dimer 53 by following the steps used in figure 1.
• the alcohol 64 is coupled with N- hydroxylaminopropanoic acid 69 to give 70.
• Alkylation of thymine with a halide 73 gives 74 which on deprotection, coupling with 76 followed by hydrolysis could afford 78.
• N-hydroxylamino propanoic aldehyde 81 is used to couple the alcohol 64.
• the dimer 88 is prepared from 83 and 86 by following the steps used in figure 8.
• thymine is alkylated with an alkylamine halide 96 (see: R. K. Olsen, K. Ramasamy and T. Emery, J. Org. Chem . , 1984, 12, 3527 and Islam et al., J. Med. Chem. , 1994, 37, 293-304 for the preparation of aminoalkyl halide) to give 97. Exposure of the compound 97 to TFA followed by alkylkation would afford 100.
• the building block 103 is obtained from 100 by dimethoxytritylation, hydrolysis, followed by
• Example 12 is an alternative route to a hydroxamate backbone dimer 111 from N-hydroxylamine 43 and an aldehyde 107 which in turn prepared from aspartic acid.
• Example 13 is an alternative route to a hydroxamate backbone dimer 111 from N-hydroxylamine 43 and an aldehyde 107 which in turn prepared from aspartic acid.
• N-hydroxylthymine is prepared (see: Kim, C.
• DMT-protected glycerol epoxide 119 provides 120.
• 1,2-dihydroxypropanoic acid 126 is coupled with N-hydroxylamine thymine 118 to give 127, which is then transformed into phophoramidite synthon 129 under standard conditions.
• the compound 118 is also coupled with adipic acid and transformed into nucleic acid building block 133.
• an aldehyde 142 and an glycine benzylester is coupled to give 143.
• Treatment of 143 with 7 should provide 145 which on exposure to acetic acid gives 148.
• Mitsunobu alkylation of 148 with Boc-NH-O-acetylhydroxylamine should give 147 which on hydrogenation the building block 150 could be obtained.
• coupling of 143 with 13 and following the same reactions as above should yield the synthon 149.
• the intermediate 177 is prepared from Boc- NH-O-benzylhydroxylamine and 175 using standard reaction conditions. Hydrogenation of 177 followed by coupling with N- hydroxythymine 116 would produce 178. Removal of the THP protecting group followed by dimethoxytritylation and
• phosphitylation should give the building block synthon 181.
• 182 could be prepared by following all the above reactions and using THP-Hydroxyacetic aldehyde instead of THP- Hydroxyacetic acid.
• the building block 191 could be prepared using the known starting material 183 and following the reaction conditions depicted at the bottom of figure 22 .
• Example 24 In Figure 24 , the starting material 200 is tranformed to the building block 207 by following the reaction conditions shown at the bottom of figure 24.
• Example 25
• Thymine acetic acid (1) Thymine (37.8 g, 300 mmol) was dissolved in a solution of potassium hydroxide (64.5 g, 1150 mmol) in 200 ml of water. While this solution was warmed in a 40°C water bath, a solution of bromoacetic acid (62.5 g, 450 mmol) in 100 mi of water was added over 1 h period. The reaction was stirred of another In at this temperature. It was allowed to cool to room temperature and the pH was adjusted to 5.5 with conc. HCl. The solution was then cooled in a
• N-Boc-L-Serine methyl ester (2) L-Serine methyl ester (15.6 g, 100 mmol) was suspended in THF/DMF(100 ml each) mixture at room temperature. To this stirred mixture was added triethylamine (11.13 g, 110 mmol) followed by di-tert-butyl dicarbonate (24.0 g, 110 mmol) and the stirring continued at room temperature for 30 minutes. Water (20 ml) was added and the solution was stirred at room temperature for 8 h. The solution was evaporated to dryness. The residue was suspended in ethyl acetate (250 ml) and treated with potassium hydrogen sulfate (0.25 N solution, 100ml).
• the product was extracted immediately with ethyl acetate solution.
• the organic extract was washed with water (100 ml), brine (100 ml) and dried over anhydrous sodium sulfate. Evaporation of the organic solvent provided an oily residue of 26g (90%).
• N-Boc-L-Serine(OTHP) methyl ester (3) The compound 2 (15 g, 68.49 mmol) was dissolved dry CH 2 Cl 2 (100 ml) and treated with 3, 4-dihydro-2H-pyran (8.4 g, 100 mmol) and catalytic amount of p-toluene sulfonic acid (100 mg) at room
• borane-methyl sulfide complex (2 M solution in THF, 100 ml 200 mmol) during 1 h period at 0°C temperature. After the addition of borane, the reaction
• N-Boc-L-Serine (OTHP) OIb (5) To a stirred solution of the compound 4 (8 g, 29.09 mmol) in dry CH 2 Cl 2 (100 ml) at 0°C was added TEA ( 3.54 g, 35 mmol) followed by isobutyryl
• N-(Thyminylacetyl)-L-Serinol(OIb) (8): Thymine acetic acid 7 (7.3 g, 40 mmol) and N-methylmorpholine (4.4 ml, 40 mmol) were dissolved in 100 mi of DMF. The solution was
• the CH 2 Cl 2 layer was dried over anhydrous Na 2 SO 4 and evaporated to dryness to give a crude product as foam.
• the crude product was purified by flash column of silica gel using CH 2 Cl 2 -> acetone containing 0.1% TEA as the eluent to give 10 g(x%) of pure product.
• the form was dried over solid NaOH in vacuum overnight.
• the form was dissolved in CH 2 Cl 2 (15 ml) and dropped into stirred solution of dry hexanes (2000 ml) under argon during 1 h period. After the addition of CH 2 Cl 2 solution, the precipitate that formed was stirred for additional 1 h and filtered, washed with dry hexanes (200 ml) and dried over soiid NaOH overnight. Yield: 9.5g (87%).
• N-(tert-butyloxycarbonyl)-O- benzyl-L-serine 2 (6.0 g, 20.34 mmol) was dissolved in dry THF and cooled to -20°C under argon atmosphere. To this cold stirred solution was added TEA (2.32 g, 23 mmol) and isobutyl chloroformate (3.13 g, 23 mmol). The stirring was continued for 30 min at -20°C under argon
• reaction mixture was filtered immediately under a blanket of argon, the precipitate was washed with dry THF (50 ml). The combined filtrate was added slowly into a cold (0°C) solution of NaBH 4 (7.4 g, 200 mmol) in THF/water (80:20, 200 ml) during 10 min period. After the addition, the reaction mixture was stirred for 2 h at 0°C and the pH
• N-(tert-Butyloaeycarbonyl)-O-Benzyl-L-Serinol-O-Ib (4): To a dried solution of N-(tert-butyloxycarbonyl)-O-benzyl-L- serinol 3 ( 4 . 3 g, 14 . 3 mmol ) in dry pyridine ( 50 ml ) was added
• reaction mixture was evaporated to dryness, dissolved in dry CH 3 OH (10 ml) and evaporated again to dryness. The residue was dried over solid KOH under vacuum for 12 h. The dried residue was used as such for further reaction without
• Thymine acetic acid 5 (2.76 g, 15 mmol) was dissolved in dry DMF (75 ml) and cooled to -20°C under argon. To this cold stirred solution was added N-methylmorpholine (1.72 g, 17 mmol) followed by isobutyl chloroformate (2.31 g, 17 mmol).
• N-(Thyminylacetyl)-L-Serinol-O-Ib (7) N-(Thyminylacetyl)-O-Benzyl- L-Serinol-O-Ib 6 (2.08 g, 5 mmol) was
• N-(Thyminylacetyl)-L-Serinol-O-Ib 7 (1.48 g, 4.5 mmol) was dissolved in dry pyridine (50 mi) under argon. To this stirred solution was added TEA (C.51 g, 5 mmol) and
• the reaction was diluted with CH 2 CH 2 (100 ml) and the organic layer was washed with 5% NaHCO 3 solution (100 ml), water (100 ml) and brine (50 ml).
• the CH 2 Cl 2 extract was dried and
• the foam was dried over solid NaOH under vacuum overnight.
• the dried foam was dissolved in dry CH 2 Cl 2 (20 ml) and dropped into a stirred solution of dry hexane (2000 ml) under argon during 1h period. After the addition, the precipitate formed was stirred for additional 1h and filtered, washed with dry hexane (100 ml) and the solid was dried over solid NaOH under vacuum for 4 h. Yield: 3.5 g (83%).
• N-(tert-Butyloxycarbonyi) - O-benzyl-D-serine 10 (7.56 g, 25.63 mmol) was dissolved in dry THF and cooled to -20°C under argon atmosphere. To this cold stirred solution was added TEA (3.03 g, 30 mmol) and isobutyl chloroformate (4.08 g, 30 mmol). The stirring was continued for 30 min at -20°C under argon
• reaction mixture was filtered immediately under a blanket of argon, the precipitate was washed with dry
• N-(tert-Butyloxycarbonyl)-O-benzyl-D-serinol 12 (6.6 g, 23.5 mmol) in dry pyridine (50 ml) was added TEA (3.03 g, 30 mmol) at room temperature.
• TEA 3.03 g, 30 mmol
• isobutyric anhydride 4.74 g, 30 mmol
• the reaction mixture was evaporated to dryness, partitioned between EtOAc (200 ml) and NaHCO 3 (5% solution, 100 ml), and extracted in EtOAc.
• the organic extract was wasned witn water (100 ml), brine (50 ml), and drie ⁇ over anhydrous Na 2 SO 4 .
• N-(tert-Butyloxycarbonyl)- O-benzyl-D-serinol-O-Ib 14 (5.0 g, 14.25 mmol) was allowed to stir at room temperature in trifluoro acetic acid (20 ml) and CH 2 Cl 2 (20 ml) for 30 min.
• the reaction mixture was evaporated to dryness, dissolved in dry CH 3 OH (10 ml) and evaporated again to dryness.
• the residue was dissolved in CH 2 Cl 2 (150 ml), the pH was adjusted to 7 with 5% NaHCO 3 solution and extracted in CH 2 Cl 2 .
• the organic layer was washed with water (50 ml) and brine (50 ml).
• the CH 2 Cl 2 extract was dried and evaporated to dryness.
• the residue that obtained was dried over solid KOH under vacuum for 12 h.
• the dried residue was used as such for further reaction without characterization.
• Thymine acetic acid 5 (2.57 g, 14 mmol) was dissolved in dry DMF (50 ml) and cooled to -20°C under argon. To this cold stirred solution was added N-methylmorpholine (1.52 g, 15 mmol) followed by isobutyl chloroformate (2.04 g, 15 mmol). After 15 min of stirring, a solution of the above amine in dry DMF (50 ml) was added into the cold stirred solution of thymine acetic acid at once. The reaction mixture was stirred at -20oC for 1 h, warmed to room temperature and the stirring continued overnight.
• Thymine acetic acid (2.2 g, 12 mmol); Isobutyl chloroformate (1.77 g, 13 mmol); N-methylmorpholine (1.52 g, 15 mmol); TFA salt (3.65 g, 10 mmol); N-methylmorpholine (1.5 g, 15 mmol) and dry DMF (100 ml). Yield: 3.5 g (84%).
• N-(Thyminylacetyl)-D-Serinol-O-Ib 16 (3.5 g, 8.39 mmol) was dissolved in ethanol (50 ml). To this solution Pd(OH) 2 (1.00 g) and cyclohexene (10 ml) were added at room temperature. The reaction mixture was heated at 70°C for 12 h. The catalyst was filtered, washed with methanol (20 ml). The filtrate was evaporated to dryness to give an white solid. Yield: 2.7 g
• N-(Thyminylacetyl)-D-Serinol-O-Ib 16 (2.7 g, 8.26 mmol) was dissolved in dry pyridine (50 ml) under argon. To this stirred solution was added TEA (1.01 g, 10 mmol) followed by
• O-Benzyl hydroxylamine hydrochloride (15.9 g, 100 mmol) was suspended in THF (150 ml) and water (50 ml) mixture. To this stirred mixture was added TEA (15.15 g, 150 mmol) followed by di-tert-butyldicarbonate (23.98 g, 110 mmol). The reaction mixture was stirred at room temperature for 12 h and
• 1-Chloro-2-(tetrahydropyranyl)oxy-ethane (29) 1-Chloro ethanol (8.06 g, 100 mmol) was dissolved in dry CH 2 Cl 2 (100 ml) and cooled to 0°C in an ice bath under argon. To this stirred solution was added dihydropyran (12.6 g, 150 mmol) followed by pyridinium -p-toluene -4-sulfonate (1.25 g, 5 mmol) and the stirring continued for overnight. The reaction mixture was evaporated to dryness and dissolved in EtOAc (200 ml).
• N-tert-butvloxvcarbonyl-O-benzylhydroxylamine 28 (5.79 g,
• Thymine acetic acid 2 (3.13 g, 17 mmol) was dissolved in dry DMF (75 ml) and cooled to -20°C under argon. To this cold stirred solution was added N-methylmorpholine (2.02 g, 20 mmol) followed by isobutyl chloroformate (2.72 g, 20 mmol).
• reaction mixture was allowed to stir at 0°C for 1 h followed by 15 h at room temperature under argon.
• the solution was cooled to °C and diluted with water (50 ml) and the pH was adjusted with AcOH to 6.
• the reaction was
• the reaction mixture was covered with aluminum foil and allowed to stir at room temperature under argon for 24 h.
• the solvent was evaporated to dryness and the residue dissolved in EtOAc (150 ml).
• the organic extract was washed with 5% NaHCO 3 solution (100 ml), water (100 ml) and brine (100 ml), and dried over anhydrous Na 2 SO 4 .
• the dried EtOAc extract was evaporated to dryness to give an orange oil.
• the crude product was purified by flash chromatography over silica gel using hexane ⁇ > EtOAc as the eluent. The fraction having the required product was pooled and evaporated to give a pale pink oil. Yield: 2.0 g (89%).
• the protecting groups were removed by treating the oligonucleotides with concentrated ammonium hydroxide at 55°C for 8 hrs.
• the oligonucleotides (DMT-on) were purified by HPLC using a reverse phase semiprep C 3 column (ABI) with a linear gradient of 5% acetonitrile in 0.1M triethylammonium acetate (buffer A) and acetonitrile (buffer B).
• the DMT protecting group was cleaved by treatment with 80% acetic acid and the product was ethanol precipitated.
• Hybridization analysis The ability of the amino acid modified oligonucleotides of the invention to hybridize to their complementary RNA and DNA sequences is determined by thermal melting analysis. The RNA complement is synthesized by
• RNA or DNA complement containing functionalized at specific locations are added to either the RNA or DNA complement at stoichiometric
• UV-Visible spectrophotometer The measurements are performed in a buffer of 10 mM Na-phosphate, pH 7.4, 0.1 mM EDTA, and
• modified pyrimidine into oligonucleotides is assessed for its effects on helix stability. Modifications that drastically alter the stability of the hybrid exhibit reductions or enhancements in the free energy (delta G) and decisions concerning their usefulness in antisense oligonucleotides are made.
• Example 33 Nuclease Resistance. Natural, phosphorothioate and modified oligonucleotides of the invention are assessed for their resistance to serum nucleases by incubation of the oligonucleotides in media containing various concentrations of fetal calf serum or adult human serum. Labeled
• oligonucleotides are incubated for various times, treated with protease K and then analyzed by gel electrophoresis on 20% polyacrylamide-urea denaturing gels and subsequent
• Autoradiography or phosphor-imaging Autoradiograms are quantitated by laser densitometry. Based upon the location of the modifications and the known length of the oligonucleotide it is possible to determine the effect of the particular modification on nuclease degradation.
• a HL60 cell line is used for the cytoplasmic nucleases.
• a post-mitochondrial supernatant is prepared by differential centrifugation and the labeled oligonucleotides are incubated in this supernatant for various times. Following the incubation, oligonucleotides are assessed for degradation as outlined above for serum nucleolytic degradation. Autoradiography results are quantitated for comparison of the unmodified i.e., phosphorothioate and the modified oligonucleotides.
• oligonucleotides showed that they are resistant to Snake Venom Phosphodiesterase.

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