We describe here the design, synthesis, physicochemical properties, and hepatitis B antiviral activity of new 2′-O-alkyl ribonucleotide N3′→P5′ phosphoramidate (2′-O-alkyl-NPO) and (thio)-phosphoramidite (2′-O-alkyl-NPS) oligonucleotide analogs. Oligonucleotides with different 2′-O-alkyl modifications such as 2′-O-methyl, -O-ethyl, -O-allyl, and -O-methoxyethyl combined with 3′-amino sugar—phosphate backbone were synthesized and evaluated. These molecules form stable duplexes with complementary DNA and RNA strands. They show an increase in duplex melting temperatures of up to 2.5°C and 4°C per linkage, respectively, compared to unmodified DNA. The results agree with predominantly C3′-endo sugar pucker conformation. Moreover, 2′-O-alkyl phosphoramidites demonstrate higher hydrolytic stability at pH 5.5 than 2′-deoxy NPOs. In addition, the relative lipophilicity of the 2′-O-alkyl-NPO and NPS oligonucleotides is higher than that of their 3′-O- counterparts. The 2′-O-alkyl-NPS oligonucleotides were evaluated as antisense (ASO) compounds in vitro and in vivo using Hepatitis B virus as a model system. Subcutaneous delivery of GalNAc conjugated 2′-O-MOE-NPS gapmers demonstrated higher activity than the 3′-O-containing 2′-O-MOE counterpart. The properties of 2′-O-alkyl-NPS constructs make them attractive candidates as ASO suitable for further evaluation and development.
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References
1.
KulkarniJ, WitzigmannD, ThomsonS, ChenS, LeavittB, CullisP and van der MeelR. (2021). The current landscape of nucleic acid therapeutics. Nat Nanotechnol, 16:630–643.
2.
KawasakiAM, CasperMD, FreierSM, LesnikEA, ZounesMC, CumminsLL, GonzalezC and CookPD. (1993). Uniformly modified 2'-deoxy-2'-fluoro phosphorothioate oligonucleotides as nuclease-resistant antisense compounds with high affinity and specificity for RNA targets. J Med Chem, 36:831–841.
3.
ShenW, De HoyosCL, SunH, VickersTA, LiangXH and CrookeST. (2018). Acute hepatotoxicity of 2’ fluoro-modified 5-10-5 gapmer phosphorothioate oligonucleotides in mice correlates with intracellular protein binding and the loss of DBHS proteins. Nucleic Acids Res, 46:2204–2217.
4.
MorozE, LeeSH, YamadaK, HalloyF, Martinez-MonteroS, JahnsH, HallJ, DamhaMJ, CastagnerB and Leroux.JC (2016). Carrier-free gene silencing by amphiphilic nucleic acid conjugates in differentiated intestinal cells. Mol Ther Nucleic Acids, 5:e364.
5.
LokCN, ViazovkinaE, MinKL, NagyE, WildsCJ, DamhaMJ and ParniakMA. (2002). Potent gene-specific inhibitory properties of mixed-backbone antisense oligonucleotides comprised of 2'-deoxy-2'-fluoro-D-arabinose and 2'-deoxyribose nucleotides. Biochemistry, 41:3457–3467.
6.
MoniaBP, LesnikEA, GonzalezC, LimaWF, McGeeD, GuinossoCJ, KawasakiAM, CookPD and FreierSM. (1993). Evaluation of 2'-modified oligonucleotides containing 2'-deoxy gaps as antisense inhibitors of gene expression. J Biol Chem, 268:14514–14522.
7.
van PuttenM, Tanganyika-de WinterC, BosgraS and Aartsma-RusA. (2019). Nonclinical exon skipping studies with 2'-O-methyl phosphorothioate antisense oligonucleotides in mdx and mdx-utrn-/- mice inspired by clinical trial results. Nucleic Acid Ther, 29:92–103.
8.
ManoharanM. (1999). 2'-carbohydrate modifications in antisense oligonucleotide therapy: importance of conformation, configuration and conjugation. Biochim Biophys Acta, 1489:117–130.
9.
ShenX and Corey.DR (2018). Chemistry, mechanism and clinical status of antisense oligonucleotides and duplex RNAs. Nucleic Acids Res, 46:1584.
10.
KosuticM, JudL, Da VeigaC, FrenerM, FausterK, KreutzC, EnnifarE and MicuraR. (2014). Surprising base pairing and structural properties of 2'-trifluoromethylthio-modified ribonucleic acids. J Am Chem Soc, 136:6656–6663.
11.
MasakiY, IriyamaY, NakajimaH, KurodaY, KanakiT, FurukawaS, SekineM and SeioK. (2018). Application of 2'-O-(2-N-methylcarbamoylethyl) nucleotides in RNase H-dependent antisense oligonucleotides. Nucleic Acid Ther, 28:307–311.
ChenJK, SchultzRG, LloydDH and GryaznovSM. (1995). Synthesis of oligodeoxyribonucleotide N3'→P5’ phosphoramidates. Nucleic Acids Res, 23:2661–2668.
14.
EgliM and GryaznovSM. (2000). Synthetic oligonucleotides as RNA mimetics: 2'-modified RNAs and N3'→P5’ phosphoramidates. Cell Mol Life Sci, 57:1440–1456.
15.
SchultzRG and GryaznovSM. (2000). Arabino-Fluorooligonucleotide N3′→P5′ phosphoramidates: synthesis and properties. Tetrahedron Lett, 41:1895–1899.
16.
SchultzRG and GryaznovSM. (1996). Oligo-2'-fluoro-2'-deoxynucleotide N3'→P5’ phosphoramidates: synthesis and properties. Nucleic Acids Res, 24:2966–2973.
17.
HerbertBS, GellertGC, HochreiterA, PongraczK, WrightWE, ZielinskaD, ChinAC, HarleyCB, ShayJW and GryaznovSM. (2005). Lipid modification of GRN163, an N3'→P5’ thio-phosphoramidate oligonucleotide, enhances the potency of telomerase inhibition. Oncogene, 24:5262–5268.
18.
DjojosubrotoMW, ChinAC, GoN, SchaetzleinS, MannsMP, GryaznovS, HarleyCB and RudolphKL. (2005). Telomerase antagonists GRN163 and GRN163L inhibit tumor growth and increase chemosensitivity of human hepatoma. Hepatology, 42:1127–1136.
19.
DikmenZG, GellertGC, JacksonS, GryaznovS, TresslerR, DoganP, WrightWE and ShayJW. (2005). In vivo inhibition of lung cancer by GRN163L: a novel human telomerase inhibitor. Cancer Res, 65:7866–7873.
20.
SteensmaDP, FenauxP, Van EygenK, RazaA, SantiniV, GermingU, FontP, Diez-CampeloM, ThepotS, VellengaE, et al. (2021). Imetelstat achieves meaningful and durable transfusion independence in high transfusion–burden patients with lower-risk myelodysplastic syndromes in a phase II study. J Clin Oncol, 39:48–56.
21.
MascarenhasJ, KomrokjiRS, PalandriF, MartinoB, NiederwieserD, ReiterA, ScottBL, BaerMR, HoffmanR, OdenikeO, et al. (2021). Randomized, single-blind, multicenter phase II study of two doses of imetelstat in relapsed or refractory myelofibrosis. J Clin Oncol, 39:2881–2892.
22.
HeidenreichO, GryaznovS and NerenbergM. (1997). RNase H-independent antisense activity of oligonucleotide N3'→P5’ phosphoramidates. Nucleic Acids Res, 25:776–780.
23.
EgliM, MinasovG, TereshkoV, PallanPS, TeplovaM, InamatiGB, LesnikEA, OwensSR, RossBS, PrakashTP, et al. (2005). Probing the influence of stereoelectronic effects on the biophysical properties of oligonucleotides: comprehensive analysis of the RNA affinity, nuclease resistance, and crystal structure of ten 2'-O-ribonucleic acid modifications. Biochemistry, 44:9045–9057.
24.
PrakashTP, Manoharan MM, KawasakiAM, FraserAS, LesnikEA, SioufiN, LeedsJM, TeplovaM and EgliM (2002). 2'-O-[2-(methylthio)ethyl]-modified oligonucleotide: An analogue of 2'-O-[2-(methoxy)-ethyl]-modified oligonucleotide with improved protein binding properties and high binding affinity to target RNA. Biochemistry, 41:11642–11648.
25.
GearyRS, Norris DD, YuR and BennettCF. (2015). Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides. Adv Drug Deliv Rev, 87:46–51.
26.
GearyRS, WatanabeTA, TruongL, FreierS, LesnikEA, SioufiNB, SasmorH, ManoharanM and LevinAA. (2001). Pharmacokinetic properties of 2'-O-(2-methoxyethyl)-modified oligonucleotide analogs in rats. J Pharmacol Exp Ther, 296:890–897.
27.
PrakashTP, MullickAE, LeeRG, YuJ, YehST, LowA, ChappellAE, OstergaardME, MurrayS, GausHJ, et al. (2019). Fatty acid conjugation enhances potency of antisense oligonucleotides in muscle. Nucleic Acids Res, 47:6029–6044.
28.
ZielinskaD, PongraczK and GryaznovS. (2006). A new approach to oligonucleotide N3′ →P5′ phosphoramidate building blocks. Tetrahedron Lett, 47:4495–4499.
Malek-AdamianE, Patrascu MBMB, JanaSK, Martinez-MonteroS, MoitessierN and DamhaMJ. (2018). Adjusting the structure of 2'-modified nucleosides and oligonucleotides via C4'-alpha-F or C4'-alpha-OMe substitution: synthesis and conformational analysis. J Org Chem, 83:9839–9849.
31.
RobertsTC, LangerR and WoodMJA. (2020). Advances in oligonucleotide drug delivery. Nat Rev Drug Discov, 19:673–694.
32.
CrookeST, WangS, VickersTA, ShenW and LiangX. (2017). Cellular uptake and trafficking of antisense oligonucleotides. Nat Biotechnol, 35:230–237.
PongraczK and GryaznovSM. (1999). Oligonucleotide N3′→P5′ thiophosphoramidates: synthesis and properties. Tetrahedron Lett, 40:7661–7664.
35.
OraM, MattilaK, Lo¨nnbergT, OivanenM and Lo¨nnbergH. (2002). Hydrolytic reactions of diribonucleoside 3′,5′-(3′-N-phosphoramidates): kinetics and mechanisms for the P − O and P − N bond cleavage of 3′-amino-3′-deoxyuridylyl-3′,5′-uridine. J Am Chem Soc, 124:14364–14372.
36.
PourshahianS and GryaznovSM. (2019). Sequencing of phosphoramidate oligonucleotides by acid hydrolysis and mass spectrometry. Anal Chem, 91:11154–11161.
37.
PourshahianS and GryaznovSM. (2021). Stereochemistry of internucleoside phosphorus atom affects sugar pucker and acid hydrolysis of N3’ - P5’ thio-phosphoramidates. Tetrahedron Lett, 68:152834–152837.
38.
CrookeST, LiangX-H, BakerBF and CrookeRM. (2021). Antisense technology: a review. J Biol Chem, 296:1–39.
39.
NairJK, WilloughbyJL, ChanA, CharisseK, AlamMR, WangQ, HoekstraM, KandasamyP, Kel'inAV, MilsteinS, et al. (2014). Multivalent N-acetylgalactosamine-conjugated siRNA localizes in hepatocytes and elicits robust RNAi-mediated gene silencing. J Am Chem Soc, 136:16958–16961.
40.
PrakashTP, GrahamMJ, YuJ, CartyR, LowA, ChappellA, SchmidtK, ZhaoC, AghajanM, MurrayHF, et al. (2014). Targeted delivery of antisense oligonucleotides to hepatocytes using triantennary N-acetyl galactosamine improves potency 10-fold in mice. Nucleic Acids Res, 42:8796–8807.
41.
PrakashTP, YuJ, MigawaMT, KinbergerGA, WanWB, OstergaardME, CartyRL, VasquezG, LowA, ChappellA, et al. (2016). Comprehensive structure-activity relationship of triantennary N-acetylgalactosamine conjugated antisense oligonucleotides for targeted delivery to hepatocytes. J Med Chem, 59:2718–2733.
42.
OstergaardME, YuJ, KinbergerGA, WanWB, MigawaMT, VasquezG, SchmidtK, GausHJ, MurrayHM, LowA, et al. (2015). Efficient synthesis and biological evaluation of 5'-GalNAc conjugated antisense oligonucleotides. Bioconjugate Chem, 26:1451–1455.
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