Classifications

• • 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 present invention relates to compounds, compositions and methods for inhibiting gene expression.
• the compounds of this invention comprise 1)
• oligonucleoside sequences of from about 6 to about 200 bases having a three atom internucleoside linkage or 2) oligonucleotide sequences of from about 9 to about 200 bases having a diol at either or both termini.
• An antisense compound is a compound that binds to or hybridizes with a nucleotide sequence in a nucleic acid, RNA or DNA, to inhibit the function or synthesis of said nucleic acid. Because of their ability to hybridize with both RNA and DNA, antisense compounds can interfere with gene expression at the level of
• Antisense molecules can be designed and synthesized to prevent the transcription of specific genes to mRNA by hybridizing with genomic DNA and directly or indirectly inhibiting the action of RNA polymerase.
• An advantage of targeting DNA is that only small amounts of antisense compounds are needed to achieve a therapeutic effect.
• antisense compounds can be designed and synthesized to hybridize with RNA to inhibit post-transcriptional modification (RNA processing) or protein synthesis (translation) mechanisms.
• RNA processing messenger RNA
• tRNA transfer RNA
• rRNA ribosomal RNA
• processing and translation mechanisms include splicing of pre-mRNA to remove introns, capping of the 5' terminus of mRNA,
• oligonucleoside sequences of antisense DNA or RNA are destroyed by exonuclease ⁇ acting at either the 5' or 3' terminus of the nucleic acid.
• exonucleases can cleave the DNA or RNA at internal phosphodiester linkages between individual nucleosides. As a result of such cleavage, the effective half-life of administered antisense compounds is very short, necessitating the use of large, frequently administered, dosages.
• Another problem is the extremely high cost of producing antisense DNA or RNA using available
• a further problem relates to the delivery of antisense agents to desired targets within the body and cell.
• Antisense agents targeted to genomic DNA must gain access to the nucleus (i.e. the agents must
• hydrophobicity must be balanced, however, against the need for aqueous solubility (increased hydrophilicity) in body fluid compartments such as the plasma and cell cytosol.
• a still further problem relates to the stability of antisense agents whether free within the body or hybridized to target nucleic acids.
• Oligonucleotide sequences such as antisense DNA are susceptible to steric reconfiguration around chiral phosphorous centers.
• the first type includes modifications to the normal internucleoside phosphodiester linkage.
• the second type includes replacement of the phosphodiester linkage with non-phosphate internucleoside linkages.
• Phosphorothioate modified phosphodiester linkages refer to phosphodiester bonds in which one or more of the bridging oxygen atoms is replaced by sulfur. Such linkages, however, are not suitable for use in antisense compounds.
• the retention of the chiral phosphorus center results in steric variation of monothioates. Further, both mono- and dithioates lack sequence specific hybridization and both are rapidly cleared from the plasma. The high affinity of
• Methyl- and ethylphosphotriesters have been prepared by reacting phosphodiester linked
• oligodeoxyribonucleotide ethylphosphotriesters is stable under normal physiological pH conditions, although it can be hydrolyzed by strong acid or base.
• Methylphosphotriesters are less stable than ethyl- and other alkylphosphotriesters at neutral pH, owing to the possibility of nucleophilic displacement of the triester methyl group by solvent. Oligodeoxyribonucleotide ethylphosphotriesters appear to be completely resistant to hydrolysis by exonucleases and are not hydrolyzed by nucleases or esterases found in fetal bovine serum or human blood serum. Uhlmann, supra.
• the methylphosphonates have several significant shortcomings in terms of therapeutic
• Oligodeoxyribonucleoside phosphoramidates have internucleoside bonds containing nitrogen-phosphorus bonds. These nucleic acid analogs can be prepared from phosphoramadite intermediates or by oxidation of
• H-phosphonate intermediates in the presence of a primary or secondary amine.
• Preparation of the H-phosphonate analogs and the oxidation reaction can be readily carried out in a commercial DNA synthesizer.
• non-ionic oligonucleoside sequences containing non-phosphate internucleoside linkages such as carbonate, acetate, carbamate and dialkyl- or diarylsilyl- derivatives have been
• Internucleoside carbamates are reported to be more water soluble than other internucleoside bridges. The utility of carbamate linkages is limited, however, since thymine carbamates do not form hybrids with complementary DNA, while cytosine carbamates do not hybridize to guanine oligomers.
• the carbamate linkage like the carbonate linkage, is stable under physiological conditions.
• the carbamate linkage does not resemble the shape of the phosphodiester internucleotide bond.
• molecular models suggest that the linkage should allow the oligomer to assume conformations which would allow it to form hydrogen-bonded complexes with complementary nucleic acids.
• a carbamate-linked oligomer containing six thymidine units does not form complexes with either A(pA) 5 or dA(pA) 5 .
• deoxycytosine units forms stable complexes with d-(pG) 6 and poly(dG).
• the internucleoside linkage of dialkyl- or diphenylsilyl oligomer analogs closely resembles the tetrahedral geometry of the normal phosphodiester internucleotide bond.
• the oligomers are prepared in solution by reacting a suitably protected nucleoside-3'-o-dialkyl- or diphenylsilyl chloride or
• trifluoromethanesulphonyl derivative with a 3'-protected nucleoside in anhydrous pyridine can be prepared by reaction of 5'-0-trityl nucleoside with dialkyl- or diphenyldichlorosilane or with the
• the present invention provides oligonucleotide analog compounds, compositions comprising such
• the present invention provides nucleotide analog compounds comprising oligonucleoside sequences of from about 6 to about 200 bases having a three atom internucleoside linkage.
• the three atom internucleoside linkage of such oligonucleoside sequences has the formula:
• each D is independently CHR, oxygen or NR 6 , wherein R is independently hydrogen, OH, SH or NH 2 , R 6 is hydrogen or C 1 -C 2 alkyl, with the proviso that only one D is oxygen or NR 6 .
• the oligonucleoside sequences comprise bases selected from the group
• compounds of the present invention comprise oligonucleoside sequences of Formula I:
• W is -D-D-D- wherein each D is independently CHR, oxygen or NR 6 , wherein R is independently hydrogen, OH,
• R 6 is hydrogen or C 1 -C 2 alkyl with the proviso that only one D is oxygen or NR 6 ;
• each W' is independently W or
• each R 1 is independently OH, SH, NR 2 R 3 wherein R 2 and R 3 are independently hydrogen or C 1 -C 6 alkyl or NHR 4 wherein
• R 4 is C 1 -C 12 acyl
• each y is independently H or OH
• each B is independently adenine, cytosine, guanine, thymine, uracil or a modification thereof;
• j is an integer from 1 to about 200;
• k is 0 or an integer from 1 to about 197; and q is o or an integer from 1 to about 197, with the proviso that the sum of j + k + q is from about 4 to about 200.
• the compounds of the present invention comprise oligonucleotide or oligonucleoside sequences optionally having a diol at either or both termini.
• Preferred diols are 1,2-diols (glycols).
• glycols are polyalkyleneglycols
• polyethyleneglycols or polypropyleneglycols preferably polyethyleneglycols or polypropyleneglycols.
• Preferred glycols are tetraethyleneglycol and
• Suitable diols may also include polyols that have all but two hydroxyls blocked.
• the compounds of the present invention are oligonucleoside sequences having a diol at either or both termini
• the compounds of the present invention have Formula II:
• R 1 is independently OH, SH, NR 2 R 3 wherein R 2 and R 3 are independently hydrogen, or C,-C 6 alkyl, or
• R 4 is C 1 -C 12 acyl
• each R 5 is independently hydrogen or C 1 -C 12 alkyl
• each of W, W', Y, B, j, k, and q is as defined above; each e and f is independently 0 to 50 with the proviso that at least one of e and f be at least 1;
• each m and n is independently 1 to 200;
• each p is independently 2 to 4.
• the sum of j + k + q is from about 9 to about 50 bases, more preferably from about 12 to about 25 and most preferably from about
• compounds of this invention comprise oligonucleotides of the formula:
• R is OH, SH, NR 2 R 3 wherein R 2 and R 3 are
• R 1 is hydrogen or C 1 -C 12 alkyl
• oligo (N) is a native or modified oligonucleotide sequence of from about 9 to about 200 bases;
• each e and f is independently 0 to 50, with the proviso that at least one of e and f be at least 1; each m and n is independently 1 to 200; and
• each p is independently 2 to 4.
• the oligonucleotide contains, in a homopolymer or heteropolymer sequence, any combination of dA, dC, dG, T.
• the compounds of this embodiment comprise oligonucleotides of the formula:
• R is OH, SH, NR 2 R 3 wherein R 2 and R 3 are
• R 4 is C 1 -C 12 acyl
• oligo N is an oligonucleotide sequence of from about 9 to about 50 bases
• e and f are independently 0 to 50, with the proviso that at least one of e and f be at least 1;
• n and n are independently 0 to 200 with the proviso that at least one of m and n be 1 to 200.
• the oligonucleotides of the present invention can include known internucleoside linking groups such as phosphodiester, silyl and other well known linking groups providing they contain an effective amount of the -D-D-D- linking groups of the present invention and/or diol terminating groups of the present invention.
• known internucleoside linking groups such as phosphodiester, silyl and other well known linking groups providing they contain an effective amount of the -D-D-D- linking groups of the present invention and/or diol terminating groups of the present invention.
• the present invention is also directed to nucleoside dimers of the formula:
• W is -D-D-D- wherein each D is independently CHR, oxygen or NR 6 wherein R is independently hydrogen, OH, SH or NH 2 , R 6 is hydrogen or C 1 -C 2 alkyl, with the proviso that only one D is oxygen or NR 6 ;
• each B is independently adenine, cytosine, guanine, thymine, uracil or a modification thereof;
• R 7 is OH, t-butyldimethylsilyloxy or a phosphoramidite and R 8 is OH, a protecting group or t-butyldimethylsilyloxy.
• the present invention further provides a method of inhibiting nuclease degradation of compounds comprising oligonucleoside sequences.
• This method comprises attaching a diol to either the 5', the 3' terminus or both termini of said compound.
• the diols are attached to the 5' and/or the 3' terminus by reacting the oligonucleotide compounds with an
• alkoxytrityldiolcyanophosphine preferably a
• dimethoxytritylglycolcyanophosphine or a
• the present invention further provides a method of inhibiting nuclease degradation of native or modified nucleotide compounds comprising preparing oligonucleoside sequences of from about 6 to about 200 bases having a three atom internucleoside linkage having the formula -D-D-D- as defined herein.
• compositions useful in inhibiting gene expression comprising compounds comprising oligonucleoside
• the compound may have a diol at either or both termini.
• Preferred diols are polyethyleneglycols.
• the present invention further provides a method of inhibiting gene expression comprising
• oligonucleoside sequence from about 6 to about 200 bases having a three atom internucleoside linkage as defined herein.
• the compounds may have a diol at either or both termini.
• Preferred diols are polyethelyeneglycols.
• Figure la depicts a synthetic pathway for preparing a nucleoside aldehyde (Compound I).
• Figure lb depicts a synthetic pathway for preparing a phosphonium iodide nucleoside (Compound II).
• Figure 2 depicts a synthetic pathway for preparing nucleoside dimers connected by a 3 carbon internucleoside linkage utilizing the aldehyde
• Figure 3 depicts a synthetic pathway for preparing a thymidine dimer utilizing a thymidine aldehyde and a phosphonium iodide thymidine (Compounds I and II respectively).
• Figure 4 depicts a synthetic pathway for preparing a nucleoside dimer connected by a two carbon-one nitrogen atom internucleoside linkage of the form 3'-C-C-N-5'. Dimers are synthesized by reacting
• nucleosides that contain amine functionalities (NH 2 ) under reductive conditions.
• Figure 5 depicts a synthetic pathway for preparing a nucleoside dimer connected by a two carbon-one nitrogen atom internucleoside linkage of the form 3'-N-C-C-5'. Dimers are synthesized by reacting
• nucleosides having aldehyde and amine functionalities under reductive conditions having aldehyde and amine functionalities under reductive conditions.
• Figure 6 depicts a synthetic pathway for preparing a thymidine dimer connected by a two carbon-one nitrogen atom internucleoside linkage of the form 3--C-C-N-5'. Dimers are synthesized by reacting
• thymidines that contain aldehydes (CHO) with thymidines that contain amine functionalities (NH 2 ) under reductive conditions.
• Figure 7 depicts a synthetic pathway for preparing a thymidine dimer connected by a two carbon-one nitrogen atom internucleoside linkage of the form 3'-N-C-C-5'. Dimers are synthesized by reacting
• the compounds of the present invention are generally oligonucleotide or oligonucleoside sequences that are resistant to nuclease degradation.
• nucleoside refers to a combination of a purine or pyrimidine base with a five-carbon sugar (pentose).
• nucleotide refers to a phosphoric acid ester of a nucleoside.
• oligonucleotide refers to polynucleotides having only phosphodiester
• internucleoside linkages e.g. "native" DNA or RNA.
• nucleosides are adenosine(A), guanosine(G), cytidine(C), uridine(U), deoxyadenosine (dA), deoxyguanosine(dG), deoxycytidine(dC) and
• the compounds of the present invention comprise oligonucleoside sequences of from about 6 to about 200 bases having a phosphodiester or a three atom internucleoside linkage.
• the three atom internucleoside linkage contains 1) three carbon atoms, 2) two carbon atoms and one oxygen atom or 3) two carbon atoms and one nitrogen atom.
• oligonucleoside sequences are sequences of native or modified nucleosides.
• internucleoside linkage refers to atoms and molecules forming a bridge between the sugar moiety carbon atom at position 3 of one native or modified nucleoside and the sugar moiety carbon atom at position 5 of an adjacent such nucleoside.
• the sugar moiety may be either a ribose or a deoxyribose moiety or an analog thereof.
• the nucleosides include A, C, G, U, dA, dC, dG, T or modifications thereof as for example 5-bromo or 5-iodouracil, 5-methyl cytosine, isocytosine (2-amino-4-oxopyrimidine), isoguanine (2-oxo-6- aminopurine), inosine (6-oxopurine), 5-vinyluracil and 5-vinylcytosine.
• the three atom internucleoside linkage has the formula:
• each D is independently CHR, oxygen or NR 6 , wherein R is independently hydrogen, OH, SH or NH 2 , oxygen, R 6 is hydrogen or C 1 -C 2 alkyl, with the proviso that only one D is oxygen or NR 6 .
• W is -D-D-D- wherein each D is independently CHR, oxygen or NR 6 , wherein R is independently hydrogen, OH, SH or NH 2 , R 6 is hydrogen or C 1 -C 2 alkyl, with the
• each W' is independently W or
• each R 1 is independently OH, SH, NR 2 R 3 wherein R 2 and R 3 are independently hydrogen or C 1 -C 6 alkyl or NHR 4 wherein R 4 is C 1 -C 12 acyl;
• each y is independently H or OH
• each B is independently adenine, cytosine, guanine, thymine, uracil or a modification thereof;
• j is an integer from 1 to about 200;
• k is o or an integer from 1 to about 197;
• q is o or an integer from 1 to about 197, with the proviso that the sum of j + k + q is from about 4 to about 200.
• the sum of j + k + q is from about 9 to about 50. In a more preferred embodiment, the sum of j + k + q is from about 12 to about 25 and, more preferably from about 15 to about 18.
• the compounds of the present invention may have a diol at either or both termini.
• Preferred diols are glycols, also known as 1,2-diols, which contain two hydroxyl groups on adjacent carbons.
• Preferred glycols are polyalkyleneglycols.
• alkylene refers to linear and branched chain radicals having 2 to 4 carbon atoms which may be optionally substituted as herein defined. Representative of such radicals are ethylene, propylene, isobutylene, and the like.
• Preferred polyalkyleneglycols are
• polyethyleneglycols such as hexaethyleneglycol and tetraethyleneglycol.
• Suitable diols may also include polyols that have all but two hydroxyls blocked.
• the diols are attached to either the 5', the 3' or both termini of the oligonucleosides via
• the diols are attached to only one terminus of an oligonucleoside sequence.
• the terminal diol is linked to a moiety selected from the group consisting of hydroxyl (OH), sulfhydryl (SH), amino (NH 2 ) , alkylamino (NH-alkyl), dialkylamino (N[alkyl] 2 ) and amido (NHfacyl]).
• the compounds of the present invention comprise oligonucleoside sequences of Formula II:
• R 1 is independently OH, SH, NHR 2 R 3 wherein R 2 and R 3 are independently hydrogen or C 1 -C 6 alkyl, or NHR 4 wherein R 4 is C 1 -C 12 acyl;
• each R 5 is independently hydrogen or C 1 -C 12 alkyl
• each of W, W', Y, B, j, k, and q is as defined above; each e and f is independently 0 to 50, with the proviso that at least one of e and f be at least 1;
• each m and n is independently 1 to 200;
• each p is independently 2 to 4.
• n are
• the sum of j + k + q is from about 9 to about 50. In a more preferred embodiment, the sum of j + k + q is from about 12 to 25, more preferably from about 15 to about 18.
• the compounds of the present invention comprise oligonucleotide sequences of from about 9 to about 200 bases having a diol at either or both termini.
• oligonucleotide sequences of from about 9 to about 200 bases having a diol at either or both termini.
• the compounds of the present invention comprise oligonucleotide sequences of from about 9 to about 200 bases having a (-D-D-D-) linkage of the present invention.
• Preferred diols are glycols, also known as 1,2-diols, which contain two hydroxyl groups on adjacent carbons.
• Preferred glycols are polyalkyleneglycols.
• alkylene refers to linear and branched chain radicals having 2 to 4 carbon atoms which may be optionally substituted as herein defined.
• radicals are ethylene, propylene, butylene and the like.
• Preferred polyalkyleneglycols are polyethyleneglycols. More preferred are
• the diols are attached to either the 5' , the 3' or both termini of the oligonucleotides via
• the diols are attached to only one terminus of an oligonucleotide sequence.
• terminal diol is linked to a moiety selected from the group consisting of hydroxyl (OH) , sulfhydryl (SH), amino (NH 2 ) , alkylamino (NH-alkyl), dialkylamino (N[alkyl] 2 ) and amido (NH[acyl]).
• OH hydroxyl
• SH sulfhydryl
• amino amino
• alkylamino NH-alkyl
• dialkylamino N[alkyl] 2
• amido NH[acyl]
• alkyl- and dialkylamino radicals include methyl-, ethyl-, propyl-, butyl-, pentyl-, hexyl-, dimethyl-, diethyl-, dipropyl-, dibutyl-, dipentyl- and dihexylamines and the like.
• Representative amido radicals include
• the compounds of the present invention comprise oligonucleotides of the formula:
• R is OH, SH, NR 2 R 3 wherein R 2 and R 3 are
• R 1 is hydrogen or C 1 -C 12 alkyl ;
• oligo (N) is a native or modified oligonucleotide sequence of from about 9 to about 200 bases;
• each e and f is independently 0 to 50;
• each m and n is independently 1 to 200;
• each p is independently 2 to 4.
• the oligonucleotide sequence is preferably a homopolymer or heteropolymer sequence containing any combination of dA, dC, dG, T or analogs thereof.
• m and n are independently 1 to 8 and, more preferably, both m and n are 4.
• Preferred oligonucleotide sequences contain from about 9 to about 50 bases, more preferably about 12 to about 25 bases, and most preferably about 15 to about 18 bases.
• the antisense compounds have polyethyalkyleneglycol at both the 5' and 3' termini and have the formula:
• R is OH, SH, NR 2 R 3 wherein R 2 and R 3 are
• R 4 is C 1 -C 12 acyl
• R 1 is hydrogen or C 1 -C 12 alkyl
• oligo (N) is a native or modified oligonucleotide sequence of from about 9 to about 200 bases;
• each e and f is independently 1 to 50;
• each m and n is independently 1 to 200;
• each p is independently 2 to 4.
• the compounds of this embodiment comprise oligonucleotides of the formula:
• R is OH, SH, NR 2 R 3 wherein R 2 and R 3 are
• R 4 is C 1 -C 12 acyl
• oligo N is an oligonucleotide sequence of from about 9 to about 50 bases.
• e, f, m and n are each independently 1 to 50.
• the oligonucleotide contains, in a homopolymer or heteropolymer sequence, any combination of dA, dC, dG, T.
• the polyethyleneglycol is tetraethyleneglycol (TEG) and both m and n are 4 or hexaethyleneglycol and both m and n are 6.
• Antisense agents hybridize with a complementary nucleotide sequence in a target nucleic acid to inhibit the translational or
• the target nucleic acid may be either RNA or DNA.
• Antisense compounds of the present invention comprise oligonucleoside sequences of from about 6 to about 200 bases having homopolymer or heteropolymer sequences comprising bases selected from the group consisting of adenine (A), cytosine (C), guanine (G) uracil (U), thymine (T) and modifications of these bases. Particular sequences are selected on the basis of their desired target. The sequence selected
• targets include the MYC oncogene, the RAS oncogene, and viral nucleic acids.
• the compounds of the present invention can be prepared by the following procedures:
• Oligonucleosides connected by a three-carbon internucleoside linkage are synthesized by reacting nucleosides having aldehyde and ylide functionalities at 3' and 6' positions respectively under Wittig
• the aldehyde (Compound I from Figure la) is synthesized from the known 3'-allyl-3'-deoxy-5'-O-tert-butyldimethylsilyl-3'-thymidine (Compound A, Figure 1).
• the allyl compound is regioselectively oxidized with a catalytic amount of osmium tetroxide and N-methylmorpholine oxide as a cooxidant.
• the resultant diol (Compound B, Figure la) is cleaved with sodium periodate to give the aldehyde in almost quantitative yield.
• a ylide is prepared from the phosphonium iodide nucleoside using potassium tert-butoxide as a base and immediately reacted with the aldehyde to give a Wittig product (Compound 1, Figure 2) in good yield.
• the Wittig product is regioselectively hydrogenated with 10% palladium on carbon (10% Pd-C) with hydrogen at atmospheric pressure in quantitative yield to saturate the double bond of the linkage.
• the saturated compound (Compound 2, Figure 2) is desilylated with
• the nucleoside dimers or higher oligomers with trialkylsilyloxy protecting groups are conjugated to form oligonucleotides of any desired length.
• the oligomers Upon completion of chain elongation, the oligomers are deprotected by standard methods.
• the terminal 5'- and 3'-hydroxyl groups of the oligomers are appropriately functionalized, respectively with tritylating reagents such as dimethoxytritylchloride and phosphoramidite.
• Oligonucleoside sequences having a two carbon- one oxygen atom internucleoside linkage are synthesized by reacting 3'-silylated, 5'-toluenesulfonyl nucleoside with a 5 '-protected nucleoside.
• a 3'-acetyl-5'-aldehyde nucleoside is prepared from a commercially available 3'-acetyl-nucleoside using standard methods well known to those of skill in the art.
• the 3 '-acetyl-5'-aldehyde nucleoside is then converted to a 3'-acetyl-5'-carbomethoxymethylene nucleoside using a modified Wittig reaction.
• the 5'-methylene side chain is reduced with sodium borohydride in alcohol, preferably isopropanol, followed by deprotection of the 3'-acetyl group with sodium methoxide in an alcohol, preferably methanol.
• the 3 '-hydroxy is then protected with a silyl group.
• the silyl group is a
• diisobutyl aluminum hydride DIBAL
• THF tetrahydrofuran
• the 5'-ethanol group is converted to a p-toluenesulfonyl group with p-toluene sulfonyl chloride in pyridine.
• the exocyclic amino group of the base moiety of the 5'-p-toluenesulfonyl nucleoside is
• a preferred protecting group for the exocyclic amino groups of adenine and cytosine is the benzoyl moiety.
• a preferred protecting group for the exocyclic amino group of guanine is the isobutyl moiety. Guanine may also be protected at the O 6 position.
• nucleoside is then reacted with a 5'-protected
• nucleoside to form a 3'-O-silyl-5'-protected nucleoside dimer with a two carbon-one oxygen atom internucleoside linkage.
• the 5'-O-protecting group is preferably a trityl and, more preferably a dimethoxytrityl.
• the 3'-O-silyl-5'-O-protected nucleoside is optionally
• nucleoside dimers are deprotected and rederivatized at the 3'-carbon atom position with a cyanophosphine reagent, preferaAbly
• nucleoside dimers or higher oligomers with trialkylsilyloxy protecting groups are conjugated to form oligonucleosides of any desired length.
• the oligomers Upon completion of chain elongation, the oligomers are deprotected by standard methods.
• the terminal 5' and 3' hydroxyl groups of the oligomers are appropriately functionalized, respectively with
• tritylating reagents such as dimethoxytritylchloride and phosphoramidite.
• Oligonucleoside sequences connected by a two carbon-one nitrogen atom internucleoside linkage of the form C-C-N are synthesized by reacting nucleosides that contain aldehydes with nucleosides that contain amine functionalities under reductive conditions as
• Both the aldehyde and the amine compounds are prepared from commercially available compounds.
• the aldehyde is prepared from 3'-allyl-3'-deoxy-5'-O-tert-butyldimethylsilyl thymidine.
• the allyl compound is regioselectively oxidized with a catalytic amount of osmium tetroxide in the presence of N-methyl
• the diol is, in turn, oxidized with sodium periodate to give the aldehyde in almost quantitative yield.
• the amine compound is synthesized from
• nucleosides In a typical procedure, the primary hydroxyl group of a nucleoside is regioselectively transformed into a tosylate group with p-toluene sulfonyl chloride and then converted into an iodide. The 3'-hydroxy of the iodide intermediate is protected with tert-butyldimethylsilyl chloride and the azido group introduced by reacting with sodium azide. The azido functionality is efficiently converted to the required amine by reduction using 10% palladium on carbon under a hydrogen atmosphere or Raney Nickel reduction conditions.
• oligonucleoside dimer with a C-C-N internucleoside linkage is formed in good yield.
• the oligonucleoside is reacted with trifluoroacetic anhydride-triethylamine, to protect the secondary aliphatic nitrogen.
• the protected oligonucleoside is desilylated with tetrabutylammonium fluoride and the primary hydroxyl group of the resultant diol is selectively protected with dimethoxytrityl chloride.
• the remaining secondary hydroxyl is transformed to the required phosphoramidite by reacting with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite.
• Oligonucleosides connected by two carbon-one nitrogen atom internucleoside linkage of the form N-C-C are synthesized by reacting nucleosides having aldehyde and amine functionalities at 3'- and 5'- positions, respectively, under reductive conditions as illustrated in Figure 5.
• the amine and the aldehyde components are synthesized from commercially available compounds.
• the amine is synthesized from 3-azido-3-deoxy thymidine (AZT).
• AZT 3-azido-3-deoxy thymidine
• the primary hydroxyl group of AZT is protected with dimethoxytritylchloride and the resultant azide regioselectively transformed to the required amine with 10% palladium on carbon in the presence of a hydrogen atmosphere or using Raney Nickel.
• the aldehyde is synthesized from commercially available 5'-O-dimethoxytritylthymidine.
• the tritylated thymidine is silylated with tert-butyldimethylsilyl chloride and the trityl group is removed under acidic conditions.
• the resultant primary hydroxyl group is oxidized under Swern conditions to give the aldehyde.
• the aldehyde is not isolated but immediately reacted with (carbethoxymethylene)triphenylphosphorane to give the unsaturated ester.
• the unsaturated ester is regioselectively hydrogenated with 10% palladium on carbon to give a saturated ester in quantitative yield.
• the saturated ester in turn is converted to the required aldehyde with diisobutyl aluminum hydride (DIBAL-H) in a highly selective manner.
• DIBAL-H diisobutyl aluminum hydride
• internucleoside linkage is obtained in good yield.
• the secondary aliphatic nitrogen of the oligonucleoside is protected with trifluoroacetic anhydride and
• oligonucleoside sequence is desilylated and the
• the nucleoside dimers or higher oligomers with trialkylsiloxyl protecting groups are conjugated to form oligonucleosides of any desired length.
• the oligomers Upon completion of chain elongation, the oligomers are desilylated by standard methods.
• the terminal 5' and 3' hydroxyl groups of the oligomers are appropriately functionalized, respectively with tritylating reagents such as dimethoxytritylchloride and phosphoramidite.
• diols are attached to either or both termini by a modification of the solid phase phosphoramidite method. Oligonucleotide Synthesis: A Practical Approach, ed. by M.J. Gait, pages 35-81, IRL Press, Washington, D.C. (1984).
• a diol is introduced at one, or both, terminal (s) of the oligonucleotide by a procedure in which the diol is reacted with an alkoxytrityl compound to form a tritylated diol.
• the diol is
• a glycol preferably a glycol, more preferably, a
• the alkoxytrityl reagent is preferably monomethoxytrityl chloride or dimethoxytrityl chloride and, most preferably dimethoxytrityl chloride.
• the tritylated diols are then reacted with a
• trityldiolcyanophosphine compound which compound is used as a phosphoramidite reagent (hereinafter referred to as a "diol phosphoramidite reagent") in the solid phase synthesis of the compounds of the present
• the initial step in solid phase synthesis is attachment of a nucleoside to a solid support
• the nucleoside is preferably attached to the CPG via a succinate linkage at the 3 '-hydroxyl position of the nucleoside.
• Other means of attaching nucleosides to solid supports are known and readily apparent to those of skill in the oligonucleotide synthesis art.
• a diol phosphoramidite reagent can be attached to the solid support prior to addition of the first nucleoside.
• the diol phosphoramidite reagent is attached to the solid support using succinate or other linkages in a manner analogous to methods used for nucleoside attachment. Means of modifying such methods for use with diol phosphoramidite reagents will be readily apparent to those of skill in the art.
• Any number of diols can be placed on the solid support prior to addition of the first nucleoside. Preferably from 1 to about 50 diols are used. Where diols are attached only to the 5' terminus, no diols are placed. on the solid support.
• chain elongation occurs via the sequential steps of removing the 5'-hydroxyl protecting group (a functionalized trityl group), activating the 5'-hydroxyl group in the presence of a phosphoramidite reagent, i.e., a 5'-trityl
• nucleoside 3'-phosphoramidite, capping the unreacted nucleosides and oxidizing the phosphorous linkage.
• the protecting group at the 5'-hydroxyl position of the attached nucleosides is removed with acid, preferably trichloroacetic acid.
• activating reagents that can be used in accordance with this method are well known to those of skill in the art.
• Preferred activating reagents are tetrazole and activator gold (Beckman Instr. Inc., Palo Alto, CA).
• the activation step occurs in the presence of the added nucleoside phosphoramidite reagent or diol phosphoramidite reagent, which latter reagent replaces the nucleoside phosphoramidite reagent of conventional synthetic methods when diol is added to the terminal (s) of the polynucleotide. Unreacted chains are terminated or capped with capping reagents such as acetic anhydride and N-methyl imidazole.
• the labile trivalent phosphorus linkage is oxidized, preferably with iodine, to the stable, pentavalent phosphodiester linkage of the
• the phosphate protecting groups are removed, the chains are separated from the solid support and the base protecting groups are removed by conventional methods. Gaits, supra at 67-70.
• the compounds of the present invention are useful in treating mammals with hereditary disorders or diseases associated with altered genetic expression mechanisms.
• attempts are underway to develop antisense therapies for use in treating viral infections such as HIV, cytomegalovirus, herpes simplex. hepatitis B, papilloma virus and picorna virus; cancers of the lung, colon, cervix, breast and ovary;
• AIDS acquired immunodeficiency syndrome
• hematological neoplasma hyperproliterative neoplasma
• compositions of the present invention useful in inhibiting gene expression comprise physiologically acceptable carriers and 1) compounds comprising
• oligonucleoside sequences of from about 6 to about 200 bases having an internucleoside linkage of the formula -D-D-D- as defined herein, optionally having a diol at either or both termini or 2) compounds comprising oligonucleotide sequences of from about 9 to about 200 bases having a diol at either or both termini.
• compositions of the present invention useful in inhibiting gene expression include one or more of the compounds of this invention formulated into compositions together with one or more non-toxic physiologically acceptable carriers, adjuvants or vehicles which are collectively referred to herein as carriers, for
• parenteral injection for oral administration in solid or liquid form, for rectal or topical administration, and the like.
• compositions can be administered to humans and animals either orally, rectally, parenterally
• compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions.
• suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
• Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
• compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents.
• adjuvants such as preserving, wetting, emulsifying, and dispensing agents.
• Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like.
• isotonic agents for example sugars, sodium chloride and the like.
• Prolonged absorption of the injectable form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
• the compounds can be incorporated into slow release or targeted delivery systems such as polymer matrices, liposomes, and microspheres. They may be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating
• compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
• Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
• the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or
• fillers or extenders as for example, starches, lactose, sucrose, glucose, mannitol and silicic acid
• binders as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose and acacia
• humectants as for example, glycerol
• disintegrating agents as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates and sodium carbonate
• solution retarders as for example paraffin
• absorption accelerators as for example, quaternary ammonium compounds
• wetting agents as for example
• lubricants as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate or mixtures thereof.
• the dosage forms may also comprise buffering agents.
• compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethyleneglycols, and the like.
• Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and others well known in this art. They may contain opacifying agents, and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner.
• embedding compositions which can be used are polymeric substances and waxes.
• the active compounds can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.
• Liquid dosage forms for oral administration include physiologically acceptable emulsions, solutions, suspensions, syrups and elixirs.
• the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl
• oils in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan or mixtures of these substances, and the like.
• composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
• adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
• compositions may contain suspending agents, as for
• microcrystalline cellulose aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
• compositions for rectal administrations are preferably suppositories which can be prepared by mixing the compounds of the present invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt in the rectum or vaginal cavity and release the active component.
• suitable non-irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt in the rectum or vaginal cavity and release the active component.
• Dosage forms for topical administration of a compound of this invention include ointments, powders, sprays and inhalants.
• the active component is admixed under sterile conditions with a physiologically
• the compounds of the present invention can also be administered in the form of liposomes.
• liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used.
• the present compositions in liposome form can contain, in addition to the lipoxygenase inhibiting compounds of the present invention, stabilizers, preservatives,
• the preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic.
• compositions of the present invention may be varied so as to obtain an amount of active ingredient that is effective to obtain a desired therapeutic response for a particular composition and method of administration.
• the selected dosage level therefore depends upon the desired therapeutic effect, on the route of
• the total daily dose of the compounds of this invention administered to a host in single or divided doses may be in amounts, for example, of from about 1 nanomol to about 5 micromols per kilogram of body weight.
• Dosage unit compositions may contain such amounts or such submultiples thereof as may be used to make up the daily dose. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the body weight, general health, sex, diet, time and route of administration, rates of absorption and excretion, combination with other drugs and the severity of the particular disease being treated.
• Dimethoxytrityl thymidine (5.0 g, 9.2 mmol) and imidazole (1.2 g, 18.4 mmol) were dissolved in 15 ml of anhydrous dimethyl formamide (DMF) and added to tert-butyldimethylsilyl chloride (1.7 g, 11.5 mmol).
• DMF dimethyl formamide
• the solution was stirred for 10 minutes followed by the addition of the vinyl thymidine, prepared according to the method of Example 4, (0.7 g, 1.9 mmol) in 5 ml of anhydrous THF.
• the reaction mixture was stirred for 45 minutes and placed in the refrigerator for 2 days.
• EXAMPLE 7 Preparation of 3'-O-t-butyldimethylsilyl- 5'-deoxy-5'-thymidyl methyl phosphonium iodide.
• EXAMPLE 8 Preparation of 5'-t-butyldimethylsilyl- 3'-deoxy-3'-(1.2"-dihydroxy-3"-propyl)- thymidine.
• Osmium tetraoxide (OsO 4 ) (4 drops, 2.5 w/v%) in butanol was added to a stirred mixture of 3'-(2"- propenyl)-3'-deoxy-5'-O-t-butyldimethylsilyl thymidine prepared according to the procedure described in J. Pro. Chem. 1989, 54:2767-2769 (C.K. Chu et al.) (183 mg, 0.5 mmol) and 4-methylmorpholine-N-oxide (53 mg, 0.45 mmol) in 5.0 ml anhydrous THF at 0oC.
• reaction mixture was then quenched with 10% aqueous sodium metabisulfite (2.0 ml), stirred for 20 minutes, filtered over a pad of silica and diluted with ethyl acetate (25.0 ml). The organic phase was washed with water (5.0 ml) and brine, and then dried with Na 2 SO 4 . The solvent was evaporated and the title compound purified by flash chromatography.
• EXAMPLE 10 preparation Of thymidine dimers with a three carbon internucleoside linkage.
• steps a-d above are used to make dimers containing three carbon internucleoside linkages in which all three carbons have the formula -CH 2 -.
• Figure 3 as follows. A drop of 2.5% (v/v) solution of osmium tetraoxide in t-butanol at 0oC was added to a stirred solution of Compound 1 and 4-methyl morpholine N-oxide (9.1 mg) in 0.8 ml of THF. The reaction mixture was kept at 0oC for 24 hours, quenched with an aqueous solution of sodium metabisulfite, diluted with ethyl acetate and washed with water and brine. The solvent was evaporated and the resulting hydroxylated dimer purified by thin layer chromatography using ethyl acetate as an eluent. The hydroxylated dimer is then protected and the 5' and 3' terminals modified as in steps b-d, above.
• EXAMPLE 11 Preparation of hvdroxvlated three carbon internucleoside linkages.
• thymidine-dimer phosphoramidite compounds produced by steps a-d were used in a modified solid phase phosphoramidite synthetic procedure to make the oligonucleoside sequences of Table 1.
• the oligodeoxynucleotides were synthesized from the 3' to the 5' terminus.
• dimethoxytrityltetraethyleneglycolcyanophosphine The activation step was followed by the capping of unreacted 5' -hydroxyl groups with acetic anhydride and N- methylimidazole. The phosphorous linkage was then oxidized with iodine in accordance with standard
• Chain elongation then proceeded via the standard sequential steps of deprotection, activation, capping and oxidation with the modification that a three-carbon linked thymidine dimer, prepared according to the methods of Examples 1-9, in the chain was added where desired during an activation step.
• the thymidine oligomers were removed from the CPG support with concentrated ammonium hydroxide. The solution was then further treated at 55oC for 8 to 15 hours to remove all the protecting groups on the exocyclic amines of the bases.
• the chilled mixture was quenched with 20 ml methanol, followed in 30 minutes with 200 ml of
• the mixture was stirred under a nitrogen atmosphere, without replenishing the ice bath, for about 20 hours.
• the title compound was further purified from a trace of starting material by chromatography on silica gel, eluting with ethyl acetate, then recrystallization from ethyl acetate/hexane to yield white crystals.
• the mixture was added to about 200 ml of ice water and extracted several times with ether.
• the combined organic extracts were washed with cold 2N hydrochloric acid, water, and brine. The washed
• reaction mixture was filtered and the solvent was evaporated to yield crude product.
• the crude product was purified by
• EXAMPLE 17 Preparation of 3'-O-t-butyldimethylsilyl- 5'-(2"-hydroxyethylene)-5'-deoxythymidine A solution of 296 mg 5 '-carbethoxymethylene- 5'-deoxythymidine was added dropwise under a nitrogen atmosphere to a cold (ice water bath), stirred solution of 205 mg imidazole and 227 mg t-butyldimethylsilyl chloride in 1 ml of anhydrous dimethylformamide. After complete addition, the mixture was removed from the ice and stirring continued at ambient temperature for two hours, then at 35oC for another two hours, and finally at 40oC for half an hour.
• the mixture was then quenched with 2 ml of methanol, followed by two to three volumes of water.
• the aqueous phase was extracted several times with ethyl acetate.
• the combined organic extracts were washed with water, saturated bicarbonate solution, and brine, dried with anhydrous magnesium sulfate, and filtered.
• the solvent was removed from the filtrate via reduced pressure to yield 0.40 g of 3'-O-t-butyldimethylsilylyl-5'-carbomethoxymethylene-5'-deoxythymidine.
• diisobutylaluminum hydride in tetrahydrofuran was added dropwise to a -30oC to -35oC chilled solution of 0.37 g of the 3'-O-t-butyldimethylsilyl-5'- carbomethoxymethylene-5'-deoxythymidine dissolved in 10 ml of anhydrous tetrahydrofuran at a temperature below -30oC.
• the reaction was stirred under a nitrogen atmosphere for an additional two hours while maintaining the internal temperature in the range of from about -30o to about -20oC.
• diisopropylethyl amine diisopropylethyl amine are added.
• the mixture is stirred for 30 minutes, followed by dropwise addition of 0.75 equivalents of 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite over a period of about 20 minutes.
• the stirring is continued for another hour, the solvent is evaporated, and the resulting
• Osmium tetraoxide (OsO 4 ) (4 drops, 2.5% w/v) in butanol was added to a stirred mixture of 3'-(2"-propenyl) -3'-deoxy-5'-O-t-butyldimethylsilyl thymidine (183 mg, 0.5 mmol), prepared according to the literature procedure, and 4-methylmorpholine-N-oxide (53 mg, 0.45 mmol) in 5.0 ml dry THF at 0oC.
• reaction mixture was then quenched with 10% aqueous sodium metabisulfite (2.0 ml), stirred for 20 minutes, filtered over a pad of silica and diluted with ethyl acetate (25.0 ml). The organic phase was washed with water (5.0 ml) and brine, and then dried with Na 2 SO 4 . The solvent was evaporated and the title compound purified by flash chromatography.
• EXAMPLE 22 Preparation of 3'-deoxy-thymid-3-yl- acetaldehyde-5'-O-t- butyldimethylsilylthymidine.
• tosylate (10.65 g, 26.9 mmol) prepared according to the method of Example 23 in dry acetone (75 ml) was added sodium iodide (10 g, 66.7 mmol) and the mixture refluxed for 16 hours. The solvent was evaporated and diluted with ethyl acetate. The organic phase was washed with water (2 ⁇ 20 ml) and brine (10 ml) and dried with sodium sulfate. The title compound was crystallized from methanol as a white crystalline solid in 90-95% yield.
• EXAMPLE 25 Preparation of 3'-O-t-butyldimethylsilyl- 5'-iodo-5'-deoxythymidine.
• EXAMPLE 26 Preparation of 3'-O-t-butyldimethylsilyl- 5'-azido-5'-deoxythymidine.
• EXAMPLE 29 Preparation of 3 '-amino-3'-deox ⁇ -5'-O- dimethoxytrityl thymidine.
• Dimethoxytrityl thymidine (5.0 g, 9.2 mmol) and imidazole (1.2 g, 18.4 mmol) were dissolved in 15 ml of anhydrous dimethyl formamide (DMF) and added to tert-butyldimethylsilyl chloride (1.7 g, 11.5 mmol). The reaction mixture was stirred for 4 hours at room temperature
• EXAMPLE 33 Preparation of 3'-O-t-butyldimethylsilyl- 5'-carbethoxy ⁇ nethyl-5'-deoxythymidine.
• EXAMPLE 36 Preparation of a deoxythymidine dimer havikg a 3'-N-C-C-5'-internucleoside linkage ( Figure 4).
• thymidine-dimer phosphoramidite compounds produced by steps a-d above were used in a modified solid phase phosphoramidite synthetic procedure to make the oligonucleoside sequences of Table 2.
• oligodeoxynucleoside sequences were synthesized from the 3' to the 5' terminus.
• the initial step was the attachment, via a 3'- succinate linkage, of a 5'-dimethoxytrityl
• Chain elongation then proceeded via the standard sequential steps of deprotection, activation, capping and oxidation with the modification that an -N- c-c- linked thymidine dimer, prepared according to the methods of Examples 30-37, was added in the chain where desired during an activation step.
• DMTCl dimethoxytrityl chloride
• DMTTEG Six grams of the DMTTEG from step (a) was admixed with 20 ml of dry dichloromethane. About 6.2 ml of Hunig's base was added to the admixture, followed by the dropwise addition of a chlorophosphine mixture to form DMTTEGCP.
• the chlorophosphine mixture was prepared by dissolving 1.67g of 2-cyanoethyl N,N-diisopropylchlorophosphoramidite in 5 ml of dry
• the oligodeoxynucleotides of Table 3 were prepared according to a modified solid phase
• A, C, G & T represent the deoxynucleotides adenylic, cytidylic, guanidylic and thymidylic acids
• adenosine nucleoside was reacted with the activating agent, tetrazole, and a phosphoramidite reagent comprising DMTTEGCP, prepared by the processes of steps a and b above.
• the activation step was followed by the capping of unreacted 5' hydroxyl groups with acetic anhydride and N-methylimidazole. The phosphorous linkage was then oxidized with iodine in accordance with standard
• the DNA strand was removed from the CPG support with concentrated ammonium hydroxide. The solution was then further treated at 55*C for 8 to 15 hours to remove all the protecting groups on the exocyclic amines of the bases.
• EXAMPLE 39 Preparation of hexaethyleneglycol (HEG)- terminated Anti-RAS oncogene DNA.
• Hexaethyleneglycol (HEG) terminated anti-RAS oncogene DNA was prepared according to the methods of Example 38. HEG was reacted with DMTCl to form DMTHEG. The DMTHEG was then reacted with a cyanophosphine compound to form DMTHEGCP, which was used in the
• A, C, G & T represent the deoxynucleotides adenylic, cytidylic, guanidylic and thymidylic acids
• the oligonucleotides of Table 4 were dissolved in water. DNA concentrations were then determined by measuring the absorbance of samples at 260 nm (on a
• oligonucleotides were incubated for 2 hours at 37oC at a total strand concentration of 6 or 7 ⁇ K in cell culture medium containing RPMI 1640; 20 mM N- (2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid), pH 7.4; and 10% fetal calf serum (FCS) (GIBCO
• FCS was heat inactivated at 56oC for 0.5 hour prior to use. Samples were then placed on ice and deproteinized using five extractions with 24:1 chloroform: isoamyl alcohol.
• Samples were either stored frozen at -20oC or
• Oligonucleotide hydrolysis was quantitated by determining the amount of disappearance of the parent compound.
• Oligonucleotides (from the reaction mixture) were separated on an LKB Ultrachrome GTi dual pump chromatography system equipped with a fixed wavelength detector (260 nm), and recording integrator, using a GenPak FAX (Waters) anion exchange column equilibrated in Buffer A (1mM EDTA; 15 mM sodium phosphate, pH 8.5). Column temperature was maintained at 60oC using a Waters column oven. Fifty microliter sample injection volumes were used. The oligonucleotides were eluted using a linear gradient of 0% to 100% Buffer B (Buffer A containing 0.5 M NaCl) over 60 minutes. Buffer flow rate was 1 mL/min.
• TEG-oligomers were resistant to hydrolysis by the FCS-associated exonucleases.
• the bis-diTEG-oligomers (7 and 10) appeared to be completely resistant to hydrolysis.
• TEG-derivatized oligodeoxynucleotides represent significant improvements over unmodified compounds in terms of resistance to exonuclease
• oligonucleotide sequences of Table 6 The percent of the population of cells in each treatment group in the S-phase of the cell cycle as compared to the nontreated control was determined using standard flow cytometric techniques. The results are shown in Table 7.
• the TEG containing antisense DNA was slightly more potent than the unmodified antisense DNA.
• exonuclease stable digonucleotides set forth in Table 9 were prepared according to the methods of Example 38.

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