Preclinical Profiling of Modified Oligonucleotides: Anticoagulation and Pharmacokinetic Properties

Abstract

Backbone, sugar and pendant-group modifications were shown to influence the anticoagulant properties of a 20-mer oligonucleotide in human plasma in vitro. The pharmacokinetics, tissue-distribution and metabolism of a chimeric oligonucleotide (CGP 69845A), which had reduced anticoagulation properties, were compared with the analogous phosphorothioate oligodeoxynucleotide (CGP 69846A) in a tumour-bearing mouse model.     

Synthesis of Triple Helix Forming Oligonucleotides Containing 2′-Deoxyformycin A

Abstract

N⁷-Benzoyl-2′-deoxyformycin A (5) was prepared from formycin A and incorporated into the triple helix forming oligonucleotide PRE2ap at CG inversion sites. The modified oligonucleotide containing three substitutions of 2′-deoxyformycin A displayed a 10-fold increase in binding affinity as compared to its unmodified counterpart. This provided a method to accommodate CG inversion sites within target sites for antiparallel triple helix formation.     

Synthesis of an Oligonucleotide Analogue of Ethenoadenosine

Abstract

A phosphoramidite building block derived from 11-carboxy-1,N ⁶-ethenoadenosine has been prepared to be used in a solid supported oligonucleotide synthesis.     

Ethyl[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-α- and β-D-erythro-Pentofuranosyl] Acetates as Versatile Intermediates in Nucleic Acid Chemistry

Abstract

The title compounds (1a,b) were synthesized in three steps from 2-deoxy-D-ribose, and used in the preparation of oligonucleotide conjugates, branched oligonucleotides as well as homo-N-nucleosides.     

Dimeric Oligonucleotide Synthons Containing 2-Deoxy-3-O-Phosphonomethyl-α-D-erythro-pentofuranosyl Nucleosides

Abstract

The synthesis of 2′-deoxy-3′-O-phosphonomethyl-α-D-erythro-pentofuranosyl-thymine and -adenine (1, B is Thy or Ade) is described. Dimeric oligonucleotide synthons such as 2 containing isosteric phosphonate internucleoside linkage were prepared.     

Nucleoside 3′-O-(2-Oxo-“Spiro”-4.4-Pentamethylene-1.3.2-Oxathiaphospholane)S: Monomers For Stereocontrolled Synthesis Of Oligo(Nucleoside Phosphorothioate/Phosphate)S

Abstract

Attempts at synthesis of “chimeric” oligonucleotide constructs (PO/PS-Oligos) possessing phosphate and P-stereodefined phosphorothioate internucleotide linkages via combined phosphoramidite/oxathiaphospholane methods were unsuccessful. Therefore, novel monomers for oxathiaphospholane method, namely 5′-O-DMT-deoxyribonucleoside 3′-O(2-oxo-.spiro-4.4-pentamethylene-1.3.2-oxathiaphospholane)s, were prepared and used together with their diastereomerically pure 2-thio analogues for the stereocontrolled synthesis of “chimeric” oligonucleotide constructs (PO/PS-Oligos).     

Synthesis of High Quality Phosphorothioate Oligonucleotides as Antisense Drugs. Use of I-Linker in the Elimination of 3′-Terminal Phosphorothioate Monoesters

Abstract

Detritylation of a 5′-O-DMT-2′-deoxyadenosine moiety attached to solid support under acidic condition leads to depurination during oligonucleotide synthesis. Deprotection followed by reversed phase HPLC purification leads to desired oligonucleotide contaminated with significant levels of 3′-terminal phosphorothiaote (3′-TPT) monoester (n?1)-mer. However, it is demonstrated that attachment of dA nucleoside through its exocyclic amino group to solid support leads to substantial reduction of 3′-TPT formation thereby improving the quality of oligonucleotide synthesized.     

Novel Base-Labile Protecting Groups for 5′-Hydroxy Function in Solid-Phase Oligonucleotide Synthesis

Abstract

The 6-(levulinyloxymethyl)-3-methoxy-2-nitrobenzoyl (LMMoNBz) and 2-(levulinyloxymethyl)-5-methoxy-4-nitrobenzoyl (LMMpNBz) groups were developed as novel base-labile protection for the 5′-hydroxy function in solid-phase oligonucleotide synthesis. A comparative study of the LMMoNBz, LMMpNBz and 2-(levulinyloxymethyl)-5-nitrobenzoyl (LMNBz) protecting groups for oligonucleotide synthesis proved strong feasibility for the LMMoNBz group.     

4′-Thio-RNA: Synthesis,Base Pairing Properties and Interaction with Dimerization Initiation Site of HIV-1

Abstract

in the present paper, we describe the synthesis of a modified 9-mer oligonucleotide, 4′-S-r(UGUGCACCU) containing for the first time 4′-thio-guanosine units. This modified 9-mer was found to inhibit in vitro genomic RNA dimerization as well as the wild type RNA.     

Synthesis and Properties of O-β-D-Ribofuranosyl-(1″-2′)-Adenosine-5″-O-Phosphate and Its Derivatives

Abstract

The synthesis of O-β-D-ribofuranosyl-(1″-2′)-adenosine-5″-O-phosphate and its suitably protected derivative for oligonucleotide synthesis have been developed.     

Synthesis of Chimerical Oligonucleotides Containing Internucleosidic Phosphodiester and S-Pivaloyl Mercaptoethyl Phosphotriester Linkages

Abstract

Novel oligonucleotide analogs that bear phosphodiester and bioreversible S-pivaloyl 2-mercaptoethyl (SPME) phosphate triester internucleosidic linkages and their thioate analogs are described. Their synthesis involves new methodology for the deprotection of base-labile oligonucleotides.     

Picomole Syntheses of High Quality Oligonucleotide Primers in Combination with the Preparation of Oligonucleotide Arrays

Abstract

The establishment of a new synthesis procedure for the preparation of oligonucleotide arrays is described. A modified phosphoramidite chemistry allowed the in situ synthesis of oligomer arrays on specially derivatized polypropylene membranes which can be used both for hybridisation experiments and for the isolation of the individual oligonucleotides.     

Nucleosides. LXII. Synthesis of 6-Methyl-8-(2-deoxy-β-D-ribofuranosyl)-isoxanthopterin and Derivatives

Abstract

The syntheses of 6-methyl-8-(2-deoxy-ß-D-ribofuranosyl)isoxanthopterin (21) and its protected 3′-O-phosphoramidite 23 were achieved from 6-methyl-2-methylthio-8-(2-deoxy-3,5-di-O-p-toluoyl-ß-D-ribofuranosyl)-3H,8H-pteridine-4,7-dione (8) in several steps. The new building block for oligonucleotide syntheses is highly fluorescent and can be considered as a substitute for 2′-deoxyguanosine.     

DNA sequence-specific ligands: XIV. Synthesis of fluorescent biologically active dimeric bisbenzimidazoles DB(3, 4, 5, 7, 11)

Five fluorescent symmetrical dimeric bisbenzimidazoles DB(n) containing four 2,6-substituted benzimidazole cores and differing in the length of the oligomethylene linker between two bisbenzimidazole rings (n = 3, 4, 5, 7, 11) have been synthesized. The ability of the dimeric bisbenzimidazoles to form complexes with the double-stranded DNA has been shown by spectral methods. Upon binding to the double-stranded DNA, DB(n) are localized in the narrow groove. The data on the inhibition of the DNA methyltransferase Dnmt3a by DB(n) indicate that dimeric bisbenzimidazoles DB(3) and DB(11) site-specifically bind to the oligonucleotide duplex.     

Oligonucleotides Containing Acyclic Nucleoside Analogues with Carbamate Internucleoside Linkages

Abstract

Synthesis of 2′-deoxy-2′,3′-secothymidine t and its dimer t?t, where the two 2′-deoxy-2′,3′-secothymidine t units are connected via a carbamate, ?=3′-NH-CO-O-5′, internucleoside linkage has been achieved. These building blocks were protected in the 5′-position, converted into their phosphoramidites, or attached onto CPG, and then used for “chimeric oligonucleotide” synthesis.     

Triplex Formation Involving 2′-O,4′-C-Methylene Bridged Nucleic Acid (2′,4′-BNA) with 2-Pyridone Base Analogue: Efficient and Selective Recognition of C:G Interruption

Abstract

For the effective recognition of C:G interruption in homopurine-homopyrimidine duplex DNA, we examined triplex-forming ability and sequence-selectivity of a triplex-forming oligonucleotide (TFO) involving of 2′-O,4′-C-methylene bridged nucleic acid with 2-pyridone base analogue. We found that the modified TFO formed stable triplex with high binding affinity and sequence-selectivity.     

Antisense Oligodeoxynucleotide Complementary to CXCR4 mRNA Block Replication of HIV-1 in COS Cells

Abstract

CXCR4 is both a chemokine receptor and an entry co-receptor for the T-cell line-adapted human immunodeficiency virus type 1 (HIV-1). To find a more efficacious therapeutic treatement of acquied immunodeficiency syndrome, we exmined the effects of antisense oligonucleotides on CXCR4 production. COS cells, stably expressing CXCR4 and CD4, were incubated with several kinds of oligonucleotides. Total human p24 antigen production was determined using an enzyme-linked immunosorbent assay system. An antisense phosphorothioate-modified oligonucleotide, complementary to the translation region of the CXCR4 mRNA, showed minimal inhibition of p24 antigen production at the high concentration of 2μM. On the other hand, the antisense phosphorothioate oligonucleotide, when used with transfection reagents, showed high efficiency at low concentrations, and confirmed the sequence-specific action. Interestingly, the oligonucleotide with the natual phosphodiester backbone, when used with the transfection reagents, also had high functional effects, comparable to the modified oligonucleotide. This defines the prerequisite criteria necessary for the design and the application of antisense oligonucleotides against HIV-1 in vivo.     

Novel Short Oligonucleotide Conjugates as Inhibitors of Human Telomerase

Abstract

A series of oligonucleotide conjugates were designed and synthesized as novel inhibitors of human telomerase. These compounds contain a relatively short (6–7-mer) oligonucleotide domain, with an N3′ → P5′ phosphoramidate (np) or thio-phosphoramidate (nps) backbone, targeted to the template region of the RNA component of the enzyme and various pendant groups attached to either their 5′- or preferably to the 3′- termini. The most potent compounds in the series inhibited telomerase with low nM IC₅₀ values in biochemical assays whereas the cognate oligonucleotides without the pendant groups were significantly less active having IC₅₀ values 100-1000-fold higher.     

Triplex Formation Involving 2′-O,4′-C-Methylene Bridged Nucleic Acid (2′,4′-BNA) with 1-Is oquinolone Base Analogue: Efficient and Selective Recognition of C:G Interruption

Abstract

For the effective recognition of C ? G interruption in homopurine-homopyrimidine duplex DNA, we examined triplex-forming ability and sequence-selectivity of a triplex-forming oligonucleotide (TFO) involving of 2′-O, 4′-C-methylene bridged nucleic acid with 1-isoquinolone base analogue. We found that the modified TFO formed stable triplex with high binding affinity and sequence-selectivity.     

Synthesis and Properties of the Oligonucleotide N3′ →P5′ Phosphoramidates

Abstract

Uniformly modified oligonucleotide N3′ → P5′ phosphoramidates were synthesized. The prepared N3′ → P5′ phosphoramidates form extremely stable duplexes and triplexes with complementary nucleic acids. Moreover, these compounds are highly resistant to enzymatic hydrolysis by snake venom phosphodiesterase and cellular nucleases and they show high antisense activity in vitro and in vivo.