The present invention relates to chirally controlled oligonucleotides, chirally controlled oligon... more The present invention relates to chirally controlled oligonucleotides, chirally controlled oligonucleotide compns., and the method of making and using the same. A chirally controlled oligonucleotide compn. comprising a plurality of oligonucleotides of at least one type, wherein each type is defined by:(1) base sequence;(2) pattern of backbone linkages;(3) pattern of backbone chiral centers; and(4) pattern of backbone X- moieties. Thus, nucleoside phosphoramidate I was prepd. and incorporated into oligonucleotides. All prepd. siRNA duplexes are evaluated in vitro for their PCSK9 gene- silencing properties, following transfection in HeLa cells or Hep3B cells.
A robust, reproducible, scalable, and automated method for the solid- phase synthesis of 5'- ... more A robust, reproducible, scalable, and automated method for the solid- phase synthesis of 5'- di- and tri- phosphates of DNA, RNA, and their chem. modified analogs that employs 5'- H- phosphonate intermediates is described. 5'- Triphosphates of oligonucleotides of varying lengths and sequences contg. different 5'- terminal nucleotides with and without internal sugar and /or backbone modifications (2'- O- Me, 2'- F, 2'- O- MOE, LNA, phosphorothioate) were efficiently prepd. as crude products or further purified by HPLC. The synthetic scheme involved the introduction of a 5'- H- phosphonate monoester onto a solid- supported oligonucleotide; the 5'- H- phosphonate monoester was further transformed into either a di- or tri- phosphate. Oligonucleotide 5'- polyphosphates were typically obtained in high yields and acceptable purity. The prepn. method was efficiently adapted to a fully automated synthesis cycle using a std. oligonucleotide synthesizer.
Although judicious use of chemical modifications has contributed to the success of nucleic acid t... more Although judicious use of chemical modifications has contributed to the success of nucleic acid therapeutics, poor systemic stability remains a major hurdle. The introduction of functional groups around the phosphate backbone can enhance the nuclease resistance of oligonucleotides (ONs). Here, we report the synthesis of enantiomerically pure (R)- and (S)-5'-C-methyl (C5'-Me) substituted nucleosides and their incorporation into ONs. These modifications generally resulted in a decrease in thermal stability of oligonucleotide (ON) duplexes in a manner dependent on the stereoconfiguration at C5' with greater destabilization characteristic of (R)-epimers. Enhanced stability against snake venom phosphodiesterase resulted from modification of the 3'-end of an ON with either (R)- or (S)-C5'-Me nucleotides. The (S)-isomers with different 2'-substituents provided greater resistance against 3'-exonucleases than the corresponding (R)-isomers. Crystal structure analyses of RNA octamers with (R)- or (S)-5'-C-methyl-2'-deoxy-2'-fluorouridine [(R)- or (S)-C5'-Me-2'-FU, respectively] revealed that the stereochemical orientation of the C5'-Me and the steric effects that emanate from the alkyl substitution are the dominant determinants of thermal stability and are likely molecular origins of resistance against nucleases. X-ray and NMR structural analyses showed that the (S)-C5'-Me epimers are spatially and structurally more similar to their natural 5' nonmethylated counterparts than the corresponding (R)-epimers.
5'-Phosphorylation is a critical step in the cascade of events that leads to loading of small... more 5'-Phosphorylation is a critical step in the cascade of events that leads to loading of small interfering RNAs (siRNAs) into the RNA-induced silencing complex (RISC) to elicit gene silencing. 5'-Phosphorylation of exogenous siRNAs is generally accomplished by a cytosolic Clp1 kinase, and in most cases, the presence of a 5'-monophosphate on synthetic siRNAs is not a prerequisite for activity. Chemically introduced, metabolically stable 5'-phosphate mimics can lead to higher metabolic stability, increased RISC loading, and higher gene silencing activities of chemically modified siRNAs,. In this study, we report the synthesis of 5'-C-malonyl RNA, a 5'-monophosphate bioisostere. A 5'-C-malonyl-modified nucleotide was incorporated at the 5'-terminus of chemically modified RNA oligonucleotides using solid-phase synthesis. In vitro silencing activity, in vitro metabolic stability, and in vitro RISC loading of 5'-C-malonyl siRNA was compared to correspond...
The invention features a oligonucleotide I, wherein Q1 is OH, O- , OR1, S- , SH, SR1; Q2 and Q3 a... more The invention features a oligonucleotide I, wherein Q1 is OH, O- , OR1, S- , SH, SR1; Q2 and Q3 are independently NH, O, S; Q4 and Q5 are independently O, CH2, CHMe, CMe2, CHF, CF2, NH, NR1, S; n is 0- 5; X and Y are independently OH, O- , OR1, SH, S- , Se, BH3, BH3- , H, N(R2) 2, alkyl, cycloalkyl, aralkyl, aryl, heteroaryl; R1 and R2 are each independently is H, alkyl, cycloalkyl, aralkyl, aryl, heteroaryl; A is absent, single or double stranded od; B is absent, linker /spacer; E is single or double stranded oligodeoxyribonucleotide, or pharmaceutically acceptable salts were prepd. and which are capable of inducing an antiviral or an antibacterial response, in particular, the induction of type I IFN, IL- 18 and /or IL- 1β by binding to RIG- 1. The invention relates to methods of making and using modified oligonucleotide comprising at least one triphosphate or analogs thereof. The invention further relates to methods for treating various disorders and diseases such as viral infecti...
The present invention provides the prepn. of nucleosides and oligonucleotides comprising a 5'... more The present invention provides the prepn. of nucleosides and oligonucleotides comprising a 5'- phosphate mimics I, wherein D1 is H, hydroxyl protecting group, solid support, phosphorus group; K is O, S, NH, N- acyl, aminoalkyl; B is H, nucleobase; V is H, substituted alkyl, substituted alkenyl, substituted alkynyl; R is H, OH, SH, halogen, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino; L is S, O, substituted amine; W1 is OH, alkoxy, oxy- aryl, oxy- heterocycle, oxy- carboxylate, substituted triazolyl, substituted amine, carbamate, oxy- sulfonyl. An aspect of the invention relates to a method of inhibiting the expression of a gene in call, the method comprising (a) contacting an oligonucleotide of the invention with the cell; and (b) maintaining the cell from step (a) for a time sufficient to obtain degrdn. of the mRNA of the target gene. Thus, nucleoside II was prepd. and used the inhibition of gene expression.
Substituted cycloalkyl and heterocyclic compds. that can be used at the 5'- ends of small RNA... more Substituted cycloalkyl and heterocyclic compds. that can be used at the 5'- ends of small RNAs, such as siRNAs, to stabilize them when used in the therapeutic inhibition of gene expression are described. These caps can include nucleotide analogs that do not contain phosphate, thiophosphate analogs, and aminoacylated analogs. Reaction schemes and syntheses are described.
Provided herein are modified 5' diphosphate nucleosides I, wherein B is nucleobase, Pg is a h... more Provided herein are modified 5' diphosphate nucleosides I, wherein B is nucleobase, Pg is a hydroxyl protecting group; M1 is H, OH, OR1; M2 is OH, OR1, NR1R2; each of R1 and R2 is independently alkyl; r is 0-1; A is O, S, CR3R4, NR5; R3 and R4 are independently H, halogen, alkyl, alkenyl, alkynyl, R5 is H, alkyl, protecting group; Q1 and Q2 are independently H, alkyl, alkenyl, alkynyl; G is H, OH, halogen, oxy-alkyl-carbonyl; and oligomeric compds. prepd. therefrom. More particularly, modified 5' diphosphate nucleosides are provided that can be further modified at the 2' and 5' positions. In some embodiments, the oligomeric compds. provided herein are expected to hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA. Thus, nucleoside II was prepd. and used in synthesis of RNA for diagnostic and inhibiting gene expression. The oligomeric compds. are also expected to be useful as primers and probes in diagnostic applications and ...
The present invention describes simple, efficient, and enzyme- free method of making oligonucleot... more The present invention describes simple, efficient, and enzyme- free method of making oligonucleotides with 5'- triphosphate. This invention presents novel process for synthesizing triphosphate oligonucleotides using: (a) a diaryl phosphonate as reagent I, wherein wherein R1 and R2 are each independently selected H, halo- alkyl, aryl, heteroaryl, cycloalkyl, heterocycle, acyl, phosphoryl, alkyl- acyl, heteroalkyl acyl, aryl- acyl or heteroaryl acyl, alkyl phosphoryl, heteroalkyl acyl, aryl phosphoryl, and heteroaryl phosphoryl, to convert the 5'- hydroxy moiety to a 5'- H- phosphonate; (b) activating the H- phosphonate of step (a) using a silylating agent, a halogenated oxidizing agent, a nitrogen- contg. heteroaryl, or a combination thereof, to form an activated H- phosphonate; and (c) treating the oligonucleotide having an activated H- phosphonate from step (b) with a poly(alkylammonium) pyrophosphate; to produce an oligonucleotide 5'- triphosphate. The process of t...
The present invention describes simple, efficient, and enzyme- free method of making oligonucleot... more The present invention describes simple, efficient, and enzyme- free method of making oligonucleotide phosphate derivs., e.g. dT7 triphosphate, comprising the steps of:(a) (a) synthesizing an oligonucleotide having a 5' hydroxyl moiety; (b) reacting the 5' hydroxyl moiety with a reagent of formula I; wherein R is each independently hydrogen, halogen, haloalkyl, halogen, NO2, CN, acyl, and sulfonyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocycle, and substituted heterocycle, and each n is 0 to 5, to convert the 5' hydroxyl moiety to a 5'- H- phosphonate; (c) activating the H- phosphonate of step (b) using a silylating agent, a halogenated oxidizing agent, a nitrogen- contg. heteroaryl, or a combination thereof, to form an activated H- phosphonate; and (d) treating the oligonucleotide having an activated H- phosphonate from step (c) with a poly(alkylammonium) phosphate salt. This invention presents novel proce...
An efficient radical deoxygenation reaction of thiocarbonylimidazolyl activated glycoside analogu... more An efficient radical deoxygenation reaction of thiocarbonylimidazolyl activated glycoside analogue using dimethyl phosphite as hydrogen source and radical chain carrier was performed as a key step in a multi step synthesis towards a common 3-deoxy glycosyl donor for 3′-deoxynucleosides. This method has safety and cost advantages compared to the generally used radical reduction reagents.
The present invention relates to chirally controlled oligonucleotides, chirally controlled oligon... more The present invention relates to chirally controlled oligonucleotides, chirally controlled oligonucleotide compns., and the method of making and using the same. A chirally controlled oligonucleotide compn. comprising a plurality of oligonucleotides of at least one type, wherein each type is defined by:(1) base sequence;(2) pattern of backbone linkages;(3) pattern of backbone chiral centers; and(4) pattern of backbone X- moieties. Thus, nucleoside phosphoramidate I was prepd. and incorporated into oligonucleotides. All prepd. siRNA duplexes are evaluated in vitro for their PCSK9 gene- silencing properties, following transfection in HeLa cells or Hep3B cells.
A robust, reproducible, scalable, and automated method for the solid- phase synthesis of 5'- ... more A robust, reproducible, scalable, and automated method for the solid- phase synthesis of 5'- di- and tri- phosphates of DNA, RNA, and their chem. modified analogs that employs 5'- H- phosphonate intermediates is described. 5'- Triphosphates of oligonucleotides of varying lengths and sequences contg. different 5'- terminal nucleotides with and without internal sugar and /or backbone modifications (2'- O- Me, 2'- F, 2'- O- MOE, LNA, phosphorothioate) were efficiently prepd. as crude products or further purified by HPLC. The synthetic scheme involved the introduction of a 5'- H- phosphonate monoester onto a solid- supported oligonucleotide; the 5'- H- phosphonate monoester was further transformed into either a di- or tri- phosphate. Oligonucleotide 5'- polyphosphates were typically obtained in high yields and acceptable purity. The prepn. method was efficiently adapted to a fully automated synthesis cycle using a std. oligonucleotide synthesizer.
Although judicious use of chemical modifications has contributed to the success of nucleic acid t... more Although judicious use of chemical modifications has contributed to the success of nucleic acid therapeutics, poor systemic stability remains a major hurdle. The introduction of functional groups around the phosphate backbone can enhance the nuclease resistance of oligonucleotides (ONs). Here, we report the synthesis of enantiomerically pure (R)- and (S)-5'-C-methyl (C5'-Me) substituted nucleosides and their incorporation into ONs. These modifications generally resulted in a decrease in thermal stability of oligonucleotide (ON) duplexes in a manner dependent on the stereoconfiguration at C5' with greater destabilization characteristic of (R)-epimers. Enhanced stability against snake venom phosphodiesterase resulted from modification of the 3'-end of an ON with either (R)- or (S)-C5'-Me nucleotides. The (S)-isomers with different 2'-substituents provided greater resistance against 3'-exonucleases than the corresponding (R)-isomers. Crystal structure analyses of RNA octamers with (R)- or (S)-5'-C-methyl-2'-deoxy-2'-fluorouridine [(R)- or (S)-C5'-Me-2'-FU, respectively] revealed that the stereochemical orientation of the C5'-Me and the steric effects that emanate from the alkyl substitution are the dominant determinants of thermal stability and are likely molecular origins of resistance against nucleases. X-ray and NMR structural analyses showed that the (S)-C5'-Me epimers are spatially and structurally more similar to their natural 5' nonmethylated counterparts than the corresponding (R)-epimers.
5'-Phosphorylation is a critical step in the cascade of events that leads to loading of small... more 5'-Phosphorylation is a critical step in the cascade of events that leads to loading of small interfering RNAs (siRNAs) into the RNA-induced silencing complex (RISC) to elicit gene silencing. 5'-Phosphorylation of exogenous siRNAs is generally accomplished by a cytosolic Clp1 kinase, and in most cases, the presence of a 5'-monophosphate on synthetic siRNAs is not a prerequisite for activity. Chemically introduced, metabolically stable 5'-phosphate mimics can lead to higher metabolic stability, increased RISC loading, and higher gene silencing activities of chemically modified siRNAs,. In this study, we report the synthesis of 5'-C-malonyl RNA, a 5'-monophosphate bioisostere. A 5'-C-malonyl-modified nucleotide was incorporated at the 5'-terminus of chemically modified RNA oligonucleotides using solid-phase synthesis. In vitro silencing activity, in vitro metabolic stability, and in vitro RISC loading of 5'-C-malonyl siRNA was compared to correspond...
The invention features a oligonucleotide I, wherein Q1 is OH, O- , OR1, S- , SH, SR1; Q2 and Q3 a... more The invention features a oligonucleotide I, wherein Q1 is OH, O- , OR1, S- , SH, SR1; Q2 and Q3 are independently NH, O, S; Q4 and Q5 are independently O, CH2, CHMe, CMe2, CHF, CF2, NH, NR1, S; n is 0- 5; X and Y are independently OH, O- , OR1, SH, S- , Se, BH3, BH3- , H, N(R2) 2, alkyl, cycloalkyl, aralkyl, aryl, heteroaryl; R1 and R2 are each independently is H, alkyl, cycloalkyl, aralkyl, aryl, heteroaryl; A is absent, single or double stranded od; B is absent, linker /spacer; E is single or double stranded oligodeoxyribonucleotide, or pharmaceutically acceptable salts were prepd. and which are capable of inducing an antiviral or an antibacterial response, in particular, the induction of type I IFN, IL- 18 and /or IL- 1β by binding to RIG- 1. The invention relates to methods of making and using modified oligonucleotide comprising at least one triphosphate or analogs thereof. The invention further relates to methods for treating various disorders and diseases such as viral infecti...
The present invention provides the prepn. of nucleosides and oligonucleotides comprising a 5'... more The present invention provides the prepn. of nucleosides and oligonucleotides comprising a 5'- phosphate mimics I, wherein D1 is H, hydroxyl protecting group, solid support, phosphorus group; K is O, S, NH, N- acyl, aminoalkyl; B is H, nucleobase; V is H, substituted alkyl, substituted alkenyl, substituted alkynyl; R is H, OH, SH, halogen, alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino; L is S, O, substituted amine; W1 is OH, alkoxy, oxy- aryl, oxy- heterocycle, oxy- carboxylate, substituted triazolyl, substituted amine, carbamate, oxy- sulfonyl. An aspect of the invention relates to a method of inhibiting the expression of a gene in call, the method comprising (a) contacting an oligonucleotide of the invention with the cell; and (b) maintaining the cell from step (a) for a time sufficient to obtain degrdn. of the mRNA of the target gene. Thus, nucleoside II was prepd. and used the inhibition of gene expression.
Substituted cycloalkyl and heterocyclic compds. that can be used at the 5'- ends of small RNA... more Substituted cycloalkyl and heterocyclic compds. that can be used at the 5'- ends of small RNAs, such as siRNAs, to stabilize them when used in the therapeutic inhibition of gene expression are described. These caps can include nucleotide analogs that do not contain phosphate, thiophosphate analogs, and aminoacylated analogs. Reaction schemes and syntheses are described.
Provided herein are modified 5' diphosphate nucleosides I, wherein B is nucleobase, Pg is a h... more Provided herein are modified 5' diphosphate nucleosides I, wherein B is nucleobase, Pg is a hydroxyl protecting group; M1 is H, OH, OR1; M2 is OH, OR1, NR1R2; each of R1 and R2 is independently alkyl; r is 0-1; A is O, S, CR3R4, NR5; R3 and R4 are independently H, halogen, alkyl, alkenyl, alkynyl, R5 is H, alkyl, protecting group; Q1 and Q2 are independently H, alkyl, alkenyl, alkynyl; G is H, OH, halogen, oxy-alkyl-carbonyl; and oligomeric compds. prepd. therefrom. More particularly, modified 5' diphosphate nucleosides are provided that can be further modified at the 2' and 5' positions. In some embodiments, the oligomeric compds. provided herein are expected to hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA. Thus, nucleoside II was prepd. and used in synthesis of RNA for diagnostic and inhibiting gene expression. The oligomeric compds. are also expected to be useful as primers and probes in diagnostic applications and ...
The present invention describes simple, efficient, and enzyme- free method of making oligonucleot... more The present invention describes simple, efficient, and enzyme- free method of making oligonucleotides with 5'- triphosphate. This invention presents novel process for synthesizing triphosphate oligonucleotides using: (a) a diaryl phosphonate as reagent I, wherein wherein R1 and R2 are each independently selected H, halo- alkyl, aryl, heteroaryl, cycloalkyl, heterocycle, acyl, phosphoryl, alkyl- acyl, heteroalkyl acyl, aryl- acyl or heteroaryl acyl, alkyl phosphoryl, heteroalkyl acyl, aryl phosphoryl, and heteroaryl phosphoryl, to convert the 5'- hydroxy moiety to a 5'- H- phosphonate; (b) activating the H- phosphonate of step (a) using a silylating agent, a halogenated oxidizing agent, a nitrogen- contg. heteroaryl, or a combination thereof, to form an activated H- phosphonate; and (c) treating the oligonucleotide having an activated H- phosphonate from step (b) with a poly(alkylammonium) pyrophosphate; to produce an oligonucleotide 5'- triphosphate. The process of t...
The present invention describes simple, efficient, and enzyme- free method of making oligonucleot... more The present invention describes simple, efficient, and enzyme- free method of making oligonucleotide phosphate derivs., e.g. dT7 triphosphate, comprising the steps of:(a) (a) synthesizing an oligonucleotide having a 5' hydroxyl moiety; (b) reacting the 5' hydroxyl moiety with a reagent of formula I; wherein R is each independently hydrogen, halogen, haloalkyl, halogen, NO2, CN, acyl, and sulfonyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocycle, and substituted heterocycle, and each n is 0 to 5, to convert the 5' hydroxyl moiety to a 5'- H- phosphonate; (c) activating the H- phosphonate of step (b) using a silylating agent, a halogenated oxidizing agent, a nitrogen- contg. heteroaryl, or a combination thereof, to form an activated H- phosphonate; and (d) treating the oligonucleotide having an activated H- phosphonate from step (c) with a poly(alkylammonium) phosphate salt. This invention presents novel proce...
An efficient radical deoxygenation reaction of thiocarbonylimidazolyl activated glycoside analogu... more An efficient radical deoxygenation reaction of thiocarbonylimidazolyl activated glycoside analogue using dimethyl phosphite as hydrogen source and radical chain carrier was performed as a key step in a multi step synthesis towards a common 3-deoxy glycosyl donor for 3′-deoxynucleosides. This method has safety and cost advantages compared to the generally used radical reduction reagents.
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Papers by Ivan Zlatev