Patent Application
20140350080
The present invention relates to modified short interfering RNA (siRNA) molecules that modulate the expression of genes via the RNA interference pathway. The nucleic acid molecules encoding the siRNAs of the invention include one or more modifications which produce differences in their physical properties when compared to wild type, unmodified siRNAs. In a preferred embodiment of the invention the nucleic acid sequences of the siRNAs include at least one nucleoside having a 2′-O-guanidinopropyl (GP) moiety. In further embodiments of the invention the modification of the siRNA results in enhanced stability of the modified siRNA, improved gene silencing by the modified siRNA and attenuated immunostimulation.
Synthetic RNAi activators have shown considerable potential for therapeutic application to silencing of pathology-causing genes. Typically these exogenous RNAi activators comprise duplex RNA of approximately 21 bp with 2 nt overhangs at the 3′ ends. To improve efficacy of siRNAs, chemical modification at the 2′-OH group of ribose has been employed. Enhanced stability, gene silencing and attenuated immunostimulation have been demonstrated using this approach. Although promising, efficient and controlled delivery of highly negatively charged nucleic acid gene silencers remains problematic.
To assess the potential utility of introducing positively charged groups at the 2′ position, our investigations aimed at assessing efficacy of novel siRNAs containing 2′-O-guanidinopropyl (GP) moieties. We describe the formation of all four GP-modified nucleosides using the synthesis sequence of Michael addition with acrylonitrile followed by Raney-Ni reduction and guanidinylation. These precursors were used successfully to generate anti-hepatitis B virus (HBV) siRNAs. Testing in a cell culture model of viral replication demonstrated that the GP modifications improved silencing. Moreover, thermodynamic stability was not affected by the GP moieties and their introduction into each position of the seed region of the siRNA guide strand did not alter the silencing efficacy of the intended HBV target. These results demonstrate that modification of siRNAs with GP groups confers properties that may be useful for advancing therapeutic application of synthetic RNAi activators.
Use of synthetic small interfering RNAs (siRNAs) to trigger RNA interference- (RNAi-) mediated gene silencing has shown considerable potential for therapeutic application [1], [2], [3]. Typically, siRNAs are synthetic mimics of natural Dicer products and comprise 21-25 nucleotide (nt) duplexes with 2 nt 3′ overhangs. Progress with use of synthetic siRNAs has profited from vast experience gained from developing antisense RNA molecules. Consequently advances have been rapid and improving siRNA efficacy has benefited from valuable biological and synthetic chemistry insights. Advantages of synthetic siRNAs over expressed RNAi activators are that they are amenable to chemical modification to improve stability, safety and specificity [4], [5]. Also, controlled large scale preparation necessary for clinical use is feasible with chemical synthetic procedures. Nevertheless, despite significant advances, the delivery of these polyanionic nucleic acids across lipid-rich cell membranes remains problematic. Vectors used to transport synthetic RNAi activators to target cells have included cationic lipid-containing lipoplexes [6], conjugations to peptides [7] or oligocationic compounds such as spermidine [8]. However, success using these methods has been variable. To overcome difficulties of the excessive negative charge of nucleic acids, while at the same time improving thermal and serum stability, we previously investigated an approach that entailed 2′-modification of ribose with cationic groups [9], [10]. Initially we generated always 2′-O-aminoethyl-adenosine and 2′-O-aminoethyl uridine. Synthesis entailed initial alkylation by methyl bromoacetate, which was followed by a series of transformation reactions. Using a luciferase reporter assay to measure knockdown, it was demonstrated that the 2′-O-aminoethyl modifications were at least as efficient as 2′-OMe siRNA modifications. An important property of the 2′-O-aminoethyl derivatives was their ability to rescue less active siRNAs when the chemical modifications were placed at the 3′ end of the siRNA passenger strand [11]. Subsequently this approach was advanced by developing methods that enabled successful alkylation of all four ribonucleosides [12]. This was achieved using phalimidoethyltriflate as an alkylating agent and with this methodology all four phosphoramidites bearing 2′-O-aminoethyl side chains were formed. Although encouraging, a problem of using these siRNA reagents is that the yields of the multistep chemical synthesis are typically low. Moreover scaling up the synthesis reaction is difficult.
To address these concerns, we have investigated utility, of an alternative 2′-O-guanidinopropyl (GP) nucleoside modification method. Using the novel approach reported here, we describe the formation of all four GP-modified nucleosides using the synthesis sequence of Michael addition with acrylonitrile [13, 14, 15] followed by Raney-Ni reduction [16] and guanidinylation. Efficiency of the GP siRNAs was assessed in a cell culture model of hepatitis B virus (HBV) replication using target sequences that have previously been shown to be suitable for RNAi-based inhibition of viral replication [17, 18, 19, 20]. Results demonstrate more effective silencing of markers of viral replication than unmodified counterparts. Moreover, the GP-modified siRNAs were more stable to serum conditions than the unmodified controls.
The present invention provides modified nucleic acid molecules and compositions comprising the modified nucleic acid molecules.
According to a first aspect of the invention the modified nucleic acid molecules comprise modified short interfering RNA (siRNA) nucleic acid molecules. The modified siRNA molecules comprise a sense strand and an antisense strand, and at least one nucleotide in the sense strand or at least one nucleotide in the antisense strand which is derived from a 2′-O-guanidinopropyl (GP) modified nucleoside. Further, the nucleic acid molecule is capable of silencing the expression of a target sequence wherein the target sequence is a DNA or RNA sequence.
The present invention teaches that at least one of the modified nucleosides is selected from the group consisting of a 2′-O-guanidinopropyl adenosine phosphoramidite, a 2′-O-guanidinopropyl cytidine phosphoramidite, a 2′-O-guanidinopropyl guanosine phosphoramidite and a 2′-O-guanidinopropyl uridine phosphoramidite. Further the invention provides for siRNAs containing combinations of the aforementioned phosphoramidites.
Preferably, the sense and antisense strands of the modified nucleic acid molecule are each, independently 18 to 26 nucleotides in length, preferably 19 to 25 nucleotides in length and most preferably 21 nucleotides in length.
Preferably, the at least one modified nucleotide may be located in the sense or the antisense stand or both. Further, the sense and antisense strands of the modified nucleic acid molecule will preferably both comprise artificially synthesised sequences.
It will be appreciated that the modified siRNA, may include an siRNA which targets DNA or RNA from any organism, including microorganisms, plants or animals. Preferably, the siRNA will target complementary nucleic acid molecules in microorganisms, including bacteria and viruses. More preferably the siRNA will target complementary nucleic acid sequence of a virus.
It will further be appreciated that the modified siRNA of the invention is a nucleic acid molecule.
In a preferred embodiment of the invention the modified siRNA inhibits viral replication. Preferably, the virus is a hepatitis virus and most preferably the virus is a hepatitis B virus.
The modified siRNA of the invention does not induce a detectable interferon response compared to an unmodified siRNA when transfected into cultured cells and/or in in vivo applications. Further, the modified siRNA has greater stability in a standard serum assay than an unmodified siRNA comprising the same sequence. In a further embodiment the modified siRNA exhibits greater knockdown of target gene expression than an unmodified siRNA comprising the same sequence.
In a preferred embodiment of the invention the antisense strand may comprise a sequence of SEQ ID NO: 1. Further the at least one 2′-O-guanidinopropyl (GP) modified nucleoside may be inserted at position 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and/or 21 of the antisense strand or at any combination of these positions.
The antisense strand may comprise an unmodified sequence of SEQ ID NO: 1 or any one of the sequences set forth in SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.
In another preferred embodiment of the invention the sense strand may comprise a sequence of SEQ ID NO: 2. Further, at least one 2′-O-guanidinopropyl (GP) modified nucleoside has been inserted at position 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and/or 21 of the sense strand or at any combination of these positions.
The sense strand may comprise an unmodified sequence of SEQ ID NOs: 2 or any one of the sequences set forth in SEQ ID NOs: 31 or 32.
According to a second aspect of the present invention there is provided for a method of treatment or prevention of viral infection, wherein the method comprises administering a therapeutically amount of the nucleic acid molecule of the invention together with and a pharmaceutically acceptable adjuvant and/or carrier to a subject in need thereof. The subject may be an animal, preferably a mammal and most preferably a human. Further, the viral infection may be hepatitis infection and most preferably the hepatitis infection is hepatitis B.
According to a third aspect of the present invention there is provided for the use of the modified siRNA of the invention in the treatment or prevention of viral infection, wherein the method comprises administering a therapeutically amount of the nucleic acid molecule of the invention together with and a pharmaceutically acceptable adjuvant and/or carrier to a subject in need thereof. The subject may be an animal, preferably a mammal and most preferably a human. Further, the viral infection may be hepatitis virus infection and most preferably the hepatitis virus infection is caused by hepatitis B.
According to a fourth aspect of the present invention there is provided for the manufacture of a medicament for use in a method of treatment or prevention of viral infection, wherein the method comprises administering a therapeutically amount of the nucleic acid molecule of the invention together with and a pharmaceutically acceptable adjuvant and/or carrier to a subject in need thereof. The subject may be an animal, preferably a mammal and most preferably a human. Further, the viral infection may be hepatitis virus infection and most preferably the hepatitis virus infection is caused by hepatitis B.
In a further aspect of the invention, there is provided for a composition comprising the siRNA of the invention together with pharmaceutically acceptable excipients, carriers, adjuvants and the like. Further, there is provided for a kit comprising the aforementioned composition together with instructions for use of the composition.
Non-limiting embodiments of the invention will now be described by way of example only and with reference to the following figures:
The present invention will now be described more fully hereinafter with reference to the accompanying figures, in which some, but not all embodiments of the invention are shown.
The invention as described should not to be limited to the specific embodiments disclosed and modifications and other embodiments are intended to be included within the scope of the invention. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
Terms used herein have their meaning recognised in the art unless otherwise indicated. According to their use here, the following terms have the meanings defined below.
As used herein the term “nucleic acid” refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses analogues of natural nucleotides that hybridise to nucleic acids in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid, sequence includes the complementary sequence thereof.
The word “nucleoside” refers to purine or pyrimidine base bound to ribose or deoxyribose sugar through a beta glycosidic link. Common examples of nucleosides are guanosine, adenosine, thymidine, cytidine and uridine.
The word “nucleotide” refers to a nucleoside that is phosphorylated on its ribose or deoxyribose moiety. The most common site of phosphorylation is at the 5′ carbon of the sugar. Nucleotide polymers form DNA or RNA. The sugar and phosphate of the polymer form the nucleic acid backbone.
“Ribose” refers to a monosaccharide found in RNA and which has the formula C5H10O5 and “deoxyribose” refers to a monosaccharide found in DNA and which has the formula C5H10O4.
The abbreviation “siRNA” refers to a “small interfering RNA”. siRNA's consist of a short double-stranded RNA molecule, the antisense- (guide) strand and the sense- (passenger) strand. Typically a siRNA molecule comprises a 19 bp duplex region with 3′ overhangs of 2 nucleotides. One strand is incorporated into a cytoplasmic RNA-induced silencing complex (RISC). This directs the sequence specific RNA cleavage that is effected by RISC. Mismatches between the siRNA guide and its target may cause translational suppression instead of RNA cleavage. siRNA may be synthetic or derived from processing of a precursor by Dicer.
As used herein “RNA interference” (RNAi) is the process by which synthetic siRNAs or the expression of a nucleic acid (including miR, siRNA, shRNA) causes sequence-specific degradation of complementary RNA, sequence-specific translational suppression or transcriptional gene silencing and further as used herein “RNAi-encoding sequence” refers to a nucleic acid sequence which, when expressed, causes RNA interference.
The word “Dicer” refers to an RNAse III enzyme, which digests double stranded RNA and is responsible for maturation of RNAi precursors. For example, Dicer is responsible for acting on pre-miRs to form mature miRs. “Drosha” is an RNase III enzyme that forms part of the nuclear microprocessor complex that recognises specific pri-miR secondary structures to cleave and release pre-miR sequences of approximately 60-80 nt.
The term “transcription” refers to the process of producing RNA from a DNA template. “In vitro transcription” refers to the process of transcription of a DNA sequence into RNA molecules using a laboratory medium which contains an RNA polymerase and RNA precursors and “intracellular transcription” refers to the transcription of a DNA sequence into RNA molecules, within a living cell. Further, “in vivo transcription” refers to the process of transcription of a DNA sequence into RNA molecules, within a living organism.
As used herein, the term ‘target nucleic acid’ or “nucleic acid target” refers to a nucleic acid sequence derived from a gene, in respect of which the RNAi-encoding sequence of the invention is designed to inhibit, block or prevent gene expression, enzymatic activity or interaction with other cellular or viral factors. In terms of the invention “target nucleic acid” or “nucleic acid target” encompass any nucleic acid capable of being targeted including without limitation including DNA, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from DNA, and also cDNA derived from RNA.
The term “guide sequence” is equivalent to the term “antisense strand” and as used herein, refers to a short single stranded RNA fragment derived from an RNAi effecter, for example siRNA, miR, shRNA that is incorporated into RISC, and which is responsible for sequence-specific degradation or translation suppression of target RNA at a target recognition sequence. Further the term “RNAi effecter” refers to any RNA sequence (e.g. shRNA, miR and siRNA) including its precursors, which can cause RNAi.
When referring to the moieties attached to the nucleosides described herein “Guanidino group” refers to a chemical moiety that includes three nitrogen atoms and one carbon atom with the chemical structure depicted below. “Propyl group” refers to a chemical moiety that includes three carbon atoms with the chemical structure depicted below and “Guanidinopropyl group” refers to a chemical moiety that comprises a guanidino group covalently linked to a propyl component.
The present invention provides nucleic acid compounds which are useful in the modulation of gene expression. The nucleic acid compounds of the invention modulate gene expression by hybridising to nucleic acid target sequences. The result of the hybridisation is the loss of normal function of the target nucleic acid. In a preferred embodiment of this invention modulation of gene expression is effected via modulation of a particular RNA associated with the particular gene-derived RNA.
The invention further provides for modulation of a target nucleic acid that is a messenger RNA. The messenger RNA is degraded by the RNA interference mechanism as well as other mechanisms in which double stranded RNA/RNA structures are recognised and degraded, cleaved or otherwise rendered inoperable.
The functions of RNA to be interfered with include replication and transcription. Replication and transcription may be from an endogenous cellular template, a vector, a plasmid construct or from other sources. The functions of RNA to be interfered with may include functions such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA.
In the context of the present invention, “modulation” and “modulation of expression” can mean either an increase (stimulation) or a decrease (inhibition) in the level or amount of a nucleic acid molecule encoding the gene, e.g., DNA or RNA. Inhibition is often the preferred form of modulation of expression and mRNA is often a preferred target nucleic acid.
The following examples are offered by way of illustration and not by way of limitation.
All reagents were of analytical reagent grade, obtained from commercial resources and used without further purification. For synthesis, solvents with quality pro analysi were used. Dry solvents were kept over molecular sieve and column chromatography technical solvents were distilled before use.
All NMR spectra were measured on Bruker AM250 (1H: 250 MHz, 13C: 63 MHz), AV300 (1H: 300 MHz, 13C: 75 MHz, 31P: 121 MHz) and AV400 (1H: 400 MHz, 13C: 101 MHz, 31P: 162 MHz) instruments. Chemical shifts (δ) are reported in parts per million (ppm). The following annotations were used with peak multiplicity: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; b, broadened. J values are given in Hz. MALDI mass spectra were recorded on a Fisons VG Tofspec spectrometer and ESI mass spectra on a Fisons VG Plattform II spectrometer. High resolution mass spectra were acquired on a Thermo MALDI Orbitrap XL.
UV-Melting curves were measured on a JASCO V-650 spectrophotometer. Melting profiles of the RNA duplexes were recorded in a phosphate buffer containing NaCl (100 mM, pH 7) at oligonucleotide concentrations 2 μM for each strand at wavelength 260 nm. Each melting curve was determined in triplicate. The temperature range was 5-95° C. with a heating rate 0.5° C. The thermodynamic data were extracted from the melting curves by means of a two state model for the transition from duplex to single strands.
Each of the four 2′-O-guanidinopropyl-nucleoside phosphoramidites was synthesised using essentially analogous methodology. The synthesis method of the adenosine (A), cytidine (C) and uridine (U) derivatives is depicted in
In the next step, the 2′-O-cyanoethyl group was transformed into a 2′-O-aminopropyl group. Reduction with hydrogen (30 bar) with Raney-nickel as catalyst in ammonia and methanol was used to achieve this according to a procedure we previously described [24]. The hydrogenation step was sensitive to reaction conditions that included the ratio of amount of starting material to catalyst, the size of the autoclave employed and reaction time. Under optimised conditions, yields from reduction of each nucleotide derivative were. moderate (about 50%). A loss of the desired product was also confirmed by the observation that part of the amino compound was not released from the catalyst during filtration, despite being subjected to several washes with methanol. To minimise losses the crude unpurified 2′4)aminopropyl compounds were used to introduce the guanidino groups. N,N′-di-Boc-N″-triflylguanidine was employed as guanidinylation agent. The procedure we employed was initially reported by Goodman et al., in 1998 [25] and is now commercially available. Our previous studies showed that the boc groups are cleaved under the repetitive deprotection conditions during oligonucleotide synthesis when employing the TBDMS-phosphoramidite method. Also, the guanidino group undergoes no side reaction during the solid phase synthesis [26].
The guanidinylation took place with good yields (70% for 3a (A), 60% for 3b (C), around 60% for 3c (U) and approximately 90% for 4c (G)). A further advantage of the synthetic procedures described here is that it is possible to introduce diversification at the 2′-O-aminopropyl site of our compounds. With common peptide-coupling reagents, such as carbodiimides and 1-hydroxybenzotriazoles, the 2′-O-aminopropyl group can readily be modified with carboxylic acid derivatives. These include amino acids, fatty acids or carboxy-modified spermine to obtain more cationic or more lipophilic oligonucleotides [24]. Also protection of the amino group with a trifluoroacetyl group during oligonucleotide solid phase synthesis would enable postsynthetic labeling with amino-reactive fluorophore derivatives (e.g. NHS-esters or isothiocyanates) or reaction with cross linkers.
After successful guanidinylation, established reaction conditions were applied to synthesize the desired phosphoramidites (1d -4d). This entailed use of protection groups that were suitable for the TBDMS method of oligoribonucleotide synthesis. The A derivative was protected with dimethylaminomethylene at the N6-position, and the exocyclic amino function of the C derivative was protected with a benzoyl group. The N2-position of the G derivative was protected with an isobutyryl group. However under the reaction conditions we employed, a mixture of the desired G derivative product as well as a compound with an additional isobutyryl group on the non-boc-protected nitrogen of the guanidino group were obtained. It was difficult to separate this additional isobutyryl group using chromatography.
However, since it would be cleaved during the ammonia deprotection step at the completion of oligonucleotide synthesis, we utilised this mixture of 4d and 4d* for solid phase oligonucleotide synthesis. To synthesise U derivatives, no further protection was necessary. For synthesis of all of the 2′-O-guanidinopropyl phosphoramidites, removal of silyl protecting groups was achieved with Et3N.3HF. The 5′-OH-group was protected with a 4,4′-dimethoxytrityl group and in a last step the 3′-OH group was converted to a phosphoramidite using 2-cyanoethyl N,N,N′,N′-tetraisopropylamino phosphane and 4,5-dicyanoimidazole as activator. Starting with the adenosine, cytidine and guanosine nucleosides, synthesis of the 2′-O-guanidinopropyl phosphoramidites took place in 10 steps and provided overall yields of 15.4% (1d), 6.3% (2d) and 7.8% (4d). Synthesis of the 2′-O-guanidinopropyl uridine phosphoramidite was performed in 8 steps with an overall yield of 11.8% (3d).
3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-N6-dimethylaminomethylene adenosine (1a) was synthesised as previously described [16].
To a solution of compound 1a (3.0 g, 5.31 mmol) in tert-butanol (25 mL), freshly distilled acrylonitrile (6.7 mL, 102 mmol) and cesium carbonate (1.6 g, 4.9 mmol) were added. The mixture was stirred vigorously at room temperature for 3 h. The reaction mixture was filtered and the residue was washed with dichloromethane. The filtrate was evaporated and the residue was purified using column chromatography with ethyl acetate/methanol (99:1-95:5, v/v) to give 3.28 g (87%) of the product. 1H NMR (400 MHz, DMSO-d6) δ [ppm] 8.90 (s, 1H, admidine-H), 8.34 (s, 1H, H2 or H8), 8.32 (s, 1H, H2 or H8), 6.02-6.01 (m, 1H, H1′), 5.05-5.01 (m, 1H, H3′), 4.64-4.62 (m, 1H, H2′), 4.08-3.84 (m, 5H, H4′, 2×H5′, O—CH2—CH2—CN), 3.20 (s, 3H, N—CH3), 3.13 (s, 3H, N—CH3), 2.83-2.80 (m, 2H, O—CH2—CH2—CN), 1.10-1.00 (m, 28H, tetraisopropyl-CH and —CH3); MS (ESI) was calculated to be 618.3 for C28H48N7O5Si2 (M+H+), and found to be 618.8.
N6-Dimethylaminomethylene-2′-O-cyanoethyl-3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-adenosine (1e) (1.0 g, 1.62 mmol) was dissolved in methanol (20 mL) then hydrazine hydrate (H2N—NH2.H2O; 500 μL, 10.3 mmol) was added. The reaction solution was stirred at room temperature for 3 h. The solvents were evaporated and the residue was purified using a silica gel column with ethylacetate as eluent to give 773 mg (87%) of 1b. 1H NMR (400 MHz, DMSO-d6) δ [ppm] 8.21 (s, 1H, H2 or H8), 8.07 (s, 1H, H2 or H8), 7.33 (bs, 2H, NH2), 5.98-5.96 (m, 1H, H1′), 5.03-4.99 (m, 1H, H3′), 4.59-4.57 (m, 1H, H2′), 4.08-3.83 (m, 5H, H4′, 2×H5′, O—CH2—CH2—CN), 2.84-2.80 (m, 2H, O—CH2—CH2—CN), 1.09-0.97 (m, 28H, tetraisopropyl-CH and —CH3); 13C NMR (101 MHz, DMSO-d6) δ [ppm] 156.01, 152.41 (C2 or C8), 148.46, 139.26 (C2 or C8), 119.20, 118.83, 87.47 (C1′), 81.11 (C2′), 80.45 (C4′), 70.04 (C3′), 65.62 H2—CN), 60.09 (C5′), 18.38 (O—CH2—CH2—CN), {17.20, 17.06, 17.05, 17.01, 16.98, 16.85, 16.81, 16.71} (tetraisopropyl-CH3), {12.60, 12.28, 12.09, 12.04} (tetraisopropyl-CH); MS (MALDI) was calculated to be 563.8 for C26H43N6O5Si2 (M+H+) and found to be 564.0.
Compound 1b (1.0 g, 1.78 mmol) was dissolved in 10 mL of methanol in a glass tube suitable for use in an autoclave. Approximately 0.5 mL of the Raney-nickel slurry was rinsed thoroughly with dry methanol and then washed into the glass tube with the solution of 1b. After addition of 5 mL methanol saturated with ammonia, the mixture was stirred for 1 h at room temperature under a hydrogen atmosphere (30 bar). The reaction mixture was filtered and the catalyst was washed several times with methanol. The filtrate was evaporated and the residue was purified using column chromatography with ethyl acetate/methanol/triethylamine (70:25:5, v/v/v) to yield 503 mg (50%) of the desired compound. When this reaction was repeated, the crude product was used for the next step without further purification. 1H NMR (400 MHz, DMSO-d6) δ [ppm] 8.20 (s, 1H, H2 or H8), 8.07 (s, 1H, H2 or H8), 7.32 (bs, 2H, NH2), 5.95-5.94 (m, 1H, H1′), 4.95-4.90 (m, 1H, H3′), 4.41-4.39 (m, 1H, H2′), 4.08-3.90 (m, 3H, H4′, 2×H5′), 3.86-3.70 (m, 2H, O—CH2—CH2—CH2—NH2), 2.66-2.61 (m, 2H, O—CH2—CH2—CH2—NH2), 1.65-1.58 (m, 2H, O—CH2—CH2—CH2—NH2), 1.08-0.96 (m, 28H, tetraisopropyl-CH and —CH3); MS (MALDI) was calculated to be 567.9 for C25H47N6O5Si2 (M+H+), and found to be 567.9.
N,N′-Di-boc-N″-triflyl guanidine (280 mg, 0.72 mmol) was dissolved in 5 mL dichloromethane then triethylamine (100 μL) was added. After cooling to 0° C., 2′-O-aminopropyl-3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-adenosine (1f) (400 mg, 0.71 mmol) was added and the mixture was stirred for 1 h at 0° C. then for 1 h at room temperature. The reaction was diluted with dichloromethane and washed with saturated sodium bicarbonate solution and brine. The organic layer was dried over Na2SO4 and the solvent was evaporated. The residue was purified using column chromatography with dichloromethane/methanol (98:2, v/v) to give a yield of 402 mg (70%) of 1c. 1H NMR (400 MHz, DMSO-d6) δ [ppm] 11.50 (s, 1H, NH-boc), 8.45-8.41 (m, 1H, NH—CH2—), 8.17 (s, 1H, H2 or H8), 8.06 (s, 1H, H2 or H8), 7.31 (bs, 2H, NH2), 6.02-5.99 (m, 1H, H1′), 4.96-4.91 (m, 1H, H3′), 4.43-4.40 (m, 1H, H2′), 4.06-3.70 (m, 5H, H4′, 2×H5′, O—CH2—CH2—CH2—NH—), 3.51-3.32 (m, 2H, O—CH2—CH2—CH2—NH—), 1.84-1.78 (m, 2H, O—CH2—CH2—CH2—NH—), 1.44 (s, 9H, C(CH3)3), 1.37 (s, 9H, C(CH3)3), 1.07-0.99 (m, 28H, tetraisopropyl-CH and —CH3); 13C NMR (101 MHz, DMSO-d6) δ [ppm] 163.00, 156.00, 155.07, 152.40 (C2 or C8), 151.96, 148.48, 139.04 (C2 or C8), 119.21, 87.69 (C1′), 82.70, 81.26 (C2′), 80.41 (C4′), 77.87, 69.99 (C3′), 69.63 (O—CH2—CH2—CH2—NH—), 60.13 (C5′), 38.41 (O—CH2—CH2—CH2—NH—), 28.71 (O—CH2—CH2—CH2—NH—), 27.85 (C(CH3)3), 27.44 (C(CH3)3), {17.19, 17.05, 17.03, 17.00, 16.95, 16.82, 16.74, 16.68} (tetraisopropyl-CH3), {12.59, 12.28, 12.09, 12.01} (tetraisopropyl-CH); MS (MALDI) was calculated to be 810.1 for O36H65N8O9Si2 (M+H+), and found to be 808.3.
Compound 1c (500 mg, 0.61 mmol) was dissolved in methanol (5 mL) and N,N-dimethylformamide dimethyl acetale (500 μL, 3.7 mmol) was added. The reaction was stirred at room temperature overnight and the solvents were evaporated. The crude product was used for further reactions without purification.
N6-Dimethylaminomethylene-2′-O—(N,N′-di-boc-guanidinopropyl)-3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-adenosine (1g) (500 mg, 0.58 mmol) was dissolved in tetrahydrofurane (5 mL) and triethylammonium trihydrofluoride (Et3N.3HF; 330 μL, 2.0 mmol) was added. The mixture was stirred at room temperature for 1.5 h, then the solvent was evaporated. The residue was purified by column chromatography with ethyl acetate/methanol (98:2-9:1, v/v) giving 300 mg (83%) of the desired product. 1H NMR (400 MHz, DMSO-d6) δ [ppm] 11.47 (s, 1H, NH-boc), 8.92 (s, 1H, N6═CH—NMe2), 8.50 (s, 1H, H2 or H8), 8.41 (s, 1H, H2 or H8), 8.33-8.29 (m, 1H, NH—CH2—), 6.11-6.09 (m, 1H, H1′), 5.20-5.24 (m, 1H, 5′-OH), 5.18-5.16 (m, 1H, 3′-OH), 4.46-4.43 (m, 1H, H2′), 4.36-4.32 (m, 1H, H3′), 4.01-3.98 (m, 1H, H4′), 3.72-3.46 (4H, 2×H5′, O—CH2—CH2—CH2—NH—), 3.33-3.28 (m, 2H, O—CH2—CH2—CH2—NH—), 3.20 (s, 3H, N—CH3), 3.13 (s, 3H, N—CH3), 1.74-1.68 (m, 2H, O—CH2—CH2—CH2—NH—), 1.45 (s, 9H, C(CH3)3), 1.37 (s, 9H, C(CH3)3); 13C NMR (101 MHz, DMSO-d6) δ [ppm] 162.97, 159.22, 157.97 (N6═CH—NMe2), 155.09, 151.89, 151.77 (C2 or C8), 151.00, 141.08 (C2 or C8), 125.70, 85.91 (C1′), 85.74 (C4′), 82.72, 81.02 (C2′), 77.99, 68.72 (C3′), 67.88 (O—CH2—CH2—CH2—NH—), 61.04 (C5′), 40.56 (N—CH3), 37.86 (O—CH2—CH2—CH2—NH—), 34.45 (N—CH3), 28.57 (O—CH2—CH2—CH2—NH—), 27.87 (C(CH3)3), 27.51 (C(CH3)3); MS (MALDI) was calculated to be 622.7 for C27H44N9O8 (M+H+), and found to be 624.6.
N6-Dimethylaminomethylene-2′-O—(N,N′-di-boc-guanidinopropyl)-adenosine (1h) (1.0 g, 1.6 mmol) was dissolved in dry pyridine (20 mL). 4,4′-Dimethoxytrityl chloride (660 mg, 1.95 mmol) was added and the reaction was stirred at room temperature overnight. The solution was diluted with dichloromethane and washed with saturated sodium bicarbonate solution. After evaporation of the solvents the residue was purified on a silica gel column with dichloromethane/methanol (98:2, v/v) containing 0.5% triethylamine, and 1.32 g (90%) of the tritylated compound was obtained. 1H NMR (400 MHz, DMSO-d6) δ [ppm] 11.48 (s, 1H, NH-boc), 8.90 (s, 1H, N6═CH—NMe2), 8.38-8.34 (m, 3H, H2, H3, NH—CH2—), 7.37-7.34 (m, 2H, DMTr), 7.27-7.17 (m, 7H, DMTr), 6.84-6.79 (m, 4H, DMTr), 6.14-6.13 (m, 1H, H1′), 5.18-5.15 (m, 1H, 3′-OH), 4.57-4.54 (m, 1H, H2′), 4.47-4.42 (m, 1H, H3′), 4.14-4.08 (m, 1H, H4′), 3.72-3.71 (m, 6H, 2×OCH3), 3.70-3.56 (m, 2H, O—CH2—CH2—CH2—NH—), 3.37-3.32 (m, 2H, O—CH2—CH2—CH2—NH—), 3.24-3.21 (m, 2H, 2×H5′), 3.19 (s, 3H, N—CH3), 3.12 (s, 3H, N—CH3), 1.77-1.70 (m, 2H, O—CH2—CH2—CH2—NH—), 1.44 (s, 9H, C(CH3)3), 1.35 (s, 9H, C(CH3)3); 13C NMR (101 MHz, DMSO-d6) δ [ppm] 162.98, 159.15, 157.97, 157.94, 157.91, 157.85 (N6═CH—NMe2), 155.09, 151.88 (C2 or C8), 151.06, 144.73, 141.18 (C2 or C8), 135.44, 135.37, {129.60, 129.56, 127.64, 127.59, 126.53} (DMTr), 125.70, 112.99 (DMTr), 86.14 (C1′), 85.34, 82.97 (C4′), 82.70, 80.36 (C2′), 77.96, 69.08 (C3′), 68.21 (O—CH2—CH2—CH2—NH—), 63.40 (C5′), 54.88 (OCH3), 40.54 (N—CH3), 37.97 (O—CH, —CH2—CH2—NH—), 34.44 (N—CH3), 28.56 (O—CH2—CH2—CH2—NH—), 27.84 (C(CH3)3), 27.50 (C(CH3)3); MS (MALDI) was calculated to be 925.1 for C48H62N6O10 (M+H+), and found to be 924.9.
N6-Dimethylaminomethylene-2′-O—(N,N′-di-boc-guanidinopropyl)-5′-O-(4,4′-dimethoxytrityl)-adenosine (1i) (320 mg, 346 μmol) was dissolved in dichloromethane (8 mL). 2-cyanoethyl N,N,N′,N′-tetraisopropylamino phosphane (132 μL, 415 μmol) and 4,5-dicyanoimidazole (47 mg, 398 μmol) were added. The mixture was stirred at room temperature. After 3 h, TLC revealed that some starting material did not react. An additional 0.6 equivalents of the phosphitylating agent as well as the catalyst were therefore added. After 4 h the reaction was complete. The mixture was diluted with dichloromethane, washed with saturated sodium bicarbonate solution and the organic layer was dried over MgSO4. The solvent was evaporated and the residue dissolved in a small amount of dichloromethane (ca. 5 mL). This solution was added dropwise into a flask with hexane (500 ml) to form a white precipitate. Two thirds of the solvent were evaporated and the remaining solvent was decanted from the solid. The precipitated product was redissolved in benzene and lyophilised to give 329 mg (84%) of *Id as a white powder. 1H NMR (300 MHz, acetone-d6) δ [ppm] 11.65 (s, 1H, NH-boc) 8.95-8.93 (m, 1H, N6═CH—NMe2), 8.42-8.27 (m, 3H, H2, H3, NH—CH2—), 7.50-7.46 (m, 2H, DMTr), 7.38-7.17 (m, 7H, DMTr), 6.87-6.80 (m, 4H, DMTr), 6.28-6.26 (m, 1H, H1′), 4.96-4.79 (m, 2H, H2′, H3′) 4.45-4.37 (m, 1H, H4′), 4.05-3.35 (m, 16H), 3.25 (s, 3H, N—CH3), 3.18 (s, 3H, N—CH3), 2.85 (m, 1H, cyanoethyl), 2.64-2.60 (m, 1H, cyanoethyl), 1.90-1.82 (m, 2H, O—CH2—CH2—CH2—NH—), 1.50-1.49 (m, 9H, C(CH3)3), 1.42-1.40 (m, 9H, C(CH3)3), 1.25-1.10 (m, 12H, iPr-CH3); 31P NMR (121 MHz, acetone-d6) δ [ppm] 149.6, 149.3; MS (ESI) was calculated to be 1125.3 for C67H79N11O11P (M+H+), and found to be 1125.7.
N4-Dimethylaminomethylene-3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-cytidine (2a) was synthesised according to a previously described procedure [29].
Compound 2a (4 g, 7.39 mmol) was dissolved in acrylonitrile (8 mL, 122 mmol) and tert-Butanol (35 mL). Cesium carbonate (1.8 g, 5.52 mmol) was added and the reaction was stirred for 2.5 h at room temperature. The mixture was filtered over celite, the solvents evaporated, and then the residue was purified using column chromatography. Ethyl acetate was initially used as solvent then changed to ethyl acetate/methanol (9:1, v/v) after the unpolar impurities had passed through the column. A yield of 3.78 g (86%) of the product were obtained. 1H NMR (400 MHz, DMSO-d6) δ [ppm] 8.62 (s, 1H, N4═CH—NMe2), 7.88 (d, 1H, J=7.3 Hz, H6), 5.90 (d, 1H, J=7.3 Hz, H5), 5.65 (s, 1H, H1′), 4.22-3.91 (m, 7H), 3.17 (s, 3H, N—CH3), 3.04 (s, 3H, N—CH3), 2.86-2.82 (m, 2H, O—CH2—CH2—CN), 1.07-0.96 (m, 28H, tetraisopropyl-CH and —CH3); 13C NMR (101 MHz, DMSO-d6) δ [ppm] 171.21 (C4), 157.77 (N4═CH—NMe2), 154.57 (C2), 140.61 (C6), 118.86 (O—CH2—CH2—CN), 101.14 (C5), 88.99 (C1′), 81.42, 80.69, 67.83, 65.22 (O—CH2—CH2—CN), 59.39 (C5′), 40.79 (N—CH3), 34.71 (N—CH3), 18.18 (O—CH2—CH2—CN), {17.22, 17.11, 17.04, 16.97, 16.84, 16.72, 16.69, 16.61} (tetraisopropyl-CH3), {12.60, 12.20, 11.88} (tetraisopropyl-CH); MS (ESI) was calculated to be 594.9 for C27H46N6O6Si2 (M+H+) and found to be 594.9.
N4-Dimethylaminomethylene-2′-O-cyanoethyl-3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-cytidine (2e) (1.0 g, 1.68 mmol) was dissolved in methanol (10 mL) and hydrazine hydrate (500 μL, 10.3 mmol) was added. The mixture was stirred for 1 h at room temperature and then the solvents were evaporated. The residue was purified on a silica gel column with ethyl acetate/methanol (95:5, v/v) to give 745 mg (82%) of 2b. 1H NMR (400 MHz, DMSO-d6) δ [ppm] 7.69 (d, 1H, J=7.4 Hz, H6), 7.21 (s, 2H, NH2), 5.69 (d, 1H, J=7.4 Hz, H5), 5.61 (s, 1H, H1′); 4.19-3.90 (m, 7H), 2.90-2.76 (m, 2H, O—CH2—CH2—CN), 1.07-0.97 (m, 28H, tetraisopropyl-CH and —CH3); 13C NMR (101 MHz, DMSO-d6) δ [ppm] 165.70, 154.60, 139.36 (C6), 118.89, 93.30 (C5), 88.66 (C1′), 81.55 (C2′), 80.49, 67.92, 65.19 (O—CH2—CH2—CN), 59.44 (C5′), 18.20 (O—CH2—CH2—CN), {17.23, 17.11, 17.05, 16.98, 16.85, 16.73, 16.72, 16.63} (tetraisopropyl-CH3), {12.62, 12.28, 12.21, 11.88} (tetraisopropyl-CH); MS (ESI) was calculated to be 539.8 for C24H43N4O6Si2 (M+H+) and found to be 540.0.
Compound 2b (500 mg, 928 μmol) was dissolved in 10 mL of methanol in a glass tube. Approximately 0.5 mL of the Raney-nickel sediment was washed thoroughly with dry methanol and was rinsed into the glass tube with the solution of 2b. After addition of 5 mL methanol saturated with ammonia, the mixture was stirred for 1 h at room temperature under a hydrogen atmosphere (30 bar). The reaction mixture was filtered through celite and the catalyst was washed several times with methanol. The solvent was evaporated and the residue was purified on a silica gel column using ethyl acetate/methanol/triethylamine (60:35:5) to give 251 mg (50%) of the product. When this procedure was repeated, the crude material after filtration and evaporation was used in further reactions without purification. 1H NMR (400 MHz, DMSO-d6) δ [ppm] 7.69 (d, 1H, J=7.2 Hz, H6), 7.18 (bs, 2H, ar. NH2), 5.68 (d, 1H, J=7.5 Hz, H5), 5.60 (s, 1H, H1′), 4.18-3.76 (m, 7H), 2.70-2.66 (m, 2H, O—CH2—CH2—CH2—NH2), 1.68-1.61 (m, 2H, O—CH2—CH2—CH2—NH2), 1.07-0.95 (m, 28H, tetraisopropyl-CH and —CH3); MS (MALDI) was calculated to be 643.8 for C24H47N4O6Si2 (M+H+) and found to be 544.6.
N,N′-Di-boc-N″-triflyl guanidine (360 mg, 920 μmol) was dissolved in 5 mL dichloromethane and triethylamine (125 μL) then added. After cooling to 0° C., 2′-O-Aminopropyl-3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-cytidine (2f) (500 mg, 922 μmol) was added and the solution was stirred for 1 h at 0° C. and then 1 h at room temperature. The reaction was diluted with dichloromethane and washed with saturated sodium bicarbonate solution and brine. The combined organic layers were dried over Na2SO4 and after evaporating the solvent the residue was purified using column chromatography with dichloromethane/methanol (98:2-95:5, v/v) to give 434 mg (60%) of 2c. 1H NMR (400 MHz, DMSO-d6) δ [ppm] 11.48 (s, 1H, NH-boc), 8.38-8.35 (m, 1H, NH—CH2—), 7.67 (d, 1H, J=7.4 Hz, H6), 7.19 (bs, 2H, NH2), 5.68 (d, 1H, J=7.4 Hz, H5), 5.63 (s, 1H, H1′), 4.17-3.78 (m, 7H), 3.49-3.33 (m, 2H, O—CH2—CH2—CH2—NH—), 1.84-1.77 (m, 2H, O—CH2—CH2—CH2—NH—), 1.45 (m, 9H, C(CH3)3), 1.38 (m, 9H, C(CH3)3), 1.06-0.96 (m, 28H, tetraisopropyl-CH and —CH3); 13C NMR (101 MHz, DMSO-d6) δ [ppm] 165.61, 162.99, 155.04, 154.52, 151.94, 139.45 (C6), 93.21 (C5), 88.97 (C1′), 82.66, 81.76 (C2′), 80.36 (C4′), 77.86, 69.11 (O—CH2—CH2—CH2—NH—), 68.27 (C3′), 59.51 (C5′), 38.28 (O—CH2—CH2—CH2—NH—), 28.61 (O—CH2—CH2—CH2—NH—), 27.86 (C(CH3)3), 27.44 (C(CH3)3), {17.22, 17.10, 17.03, 16.96, 16.83, 16.70, 16.68, 16.60} (tetraisopropyl-CH3), {12.59, 12.26, 12.21, 11.87} (tetraisopropyl-CH); MS (MALDI) was calculated to be 786.1 for C36H65N6O10Si2 (M+H+) and found to be 786.4.
Compound 2c (1.0 g, 1.27 mmol) was dissolved in dry pyridine (10 mL) and the solution was cooled in an ice bath. Benzoyl chloride (240 μL, 2.06 mmol) was added and the reaction solution was stirred at 0° C. for 1 h. The reaction was quenched with water and ammonia (25% in water; 3 mL) was added. The mixture was then stirred for 30 minutes at room temperature. The solvents were evaporated and the residue was dissolved in dichloromethane and washed with saturated sodium bicarbonate solution. The organic layer was dried over Na2SO4 and after evaporating the solvent, the residue was purified by column chromatography using dichloromethane/methanol (98:2, v/v) and 950 mg (84%) of the product were obtained. 1H NMR (400 MHz, DMSO-d6) δ [ppm] 11.50 (s, 1H, NH), 11.31 (s, 1H, NH), 8.40-8.37 (m, 1H, NH—CH2—), 8.15 (d, 1H, J=7.3 Hz, H6), 8.03-7.99 (m, 2H, benzoyl), 7.65-7.60 (m, 1H, benzoyl), 7.53-7.49 (m, 2H, benzoyl), 7.38 (d, 1H, J=7.3 Hz, H5), 5.73 (s, 1H, H1′), 4.24-4.13 (m, 3H, H3′, H4′, H5′), 4.02-4.03 (m, 1H, H2′), 3.97-3.92 (m, 1H, H5′), 3.87-3.83 (m, 2H, O—CH2—CH2—CH2—NH—), 3.52-3.35 (m, 2H, O—CH2—CH2—CH2—NH—), 1.87-1.80 (m, 2H, O—CH2—CH2—CH2—NH—), 1.45 (m, 9H, C(CH3)3), 1.38 (m, 9H, C(CH3)3), 1.08-0.95 (m, 28H, tetraisopropyl-CH and —CH3); 13C NMR (101 MHz, DMSO-d6) J[ppm] 167.21, 163.14, 163.00, 155.06, 154.01, 151.97, 143.37 (C6), 132.97, 132.64, 128.31, 95.61 (C5), 89.46 (C1′), 82.67, 81.30 (C2′), 80.86 (C4′), 77.86, 69.26 (O—CH2—CH2—CH2—NH—), 67.95 (C3′), 59.38 (C5′), 38.28 (O—CH2—CH2—CH2—NH—), 28.62 (O—CH2—CH2—CH2—NH—), 27.86 (C(CH3)3), 27.43 (C(CH3)3), {17.22, 17.11, 17.04, 16.97, 16.89, 16.73, 16.71, 16.66} (tetraisopropyl-CH3), {12.56, 12.29, 12.22, 11.86} (tetraisopropyl-CH); MS (ESI) was calculated to be 890.2 for C42H69N6O11Si2 (M+H+), and found to be 890.4.
N4-Benzoyl-2′-O—(N,N′-di-boc-guanidinopropyl)-3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-cytidine (2g) (900 mg, 1.01 mmol) was dissolved in tetrahydrofurane (20 mL). Triethylamine trihydrofluoride (Et3N.3HF; 560 μL, 3.54 mmol) was added and the solution was stirred at room temperature for 2 h. The solvent was evaporated and the residue was purified using column chromatography with dichloromethane/methanol (98:2-97:3, v/v) to give 607 mg (93%) of the product as a pale yellow foam. 1H NMR (300 MHz, DMSO-d6) δ [ppm] 11.50 (s, 1H, NH), 11.28 (bs, 1H, NH), 8.57 (d, 1H, J=7.5 Hz, H6), 8.40-8.35 (m, 1H, NH—CH2—), 8.02-7.98 (m, 2H, benzoyl), 7.66-7.60 (m, 1H, benzoyl), 7.54-7.48 (m, 2H, benzoyl), 7.34 (d, 1H, J=7.2 Hz, H5), 5.86-5.85 (m, 1H, H1′), 5.24 (t, 1H, J=5.0 Hz, 5′-OH), 4.98 (d, 1H, J=6.8 Hz, 3′-OH), 4.12-3.60 (m, 7H), 3.44-3.37 (m, 2H, O—CH2—CH2—CH2—NH—), 1.85-1.76 (m, 2H, O—CH2—CH2—CH2—NH—), 1.46 (m, 9H, (C(CH3)3), 1.38 (m, 9H, C(CH3)3); 13C NMR (75 MHz, acetone-d6) δ [ppm] 169.22, 165.59, 164.82, 157.83, 156.15, 154.80, 147.10 (C6), 135.58, 134.60, 130.46, 130.05, 97.67 (C5), 91.39 (C1′), 86.19 (C4′), 84.69 (C(CH3)3), 84.62 (C2′), 79.88 (C(CH3)3), 70.71 (O—CH2—CH2—CH2—NH—), 69.63 (C3′), 61.46 (C5′), 40.29 (O—CH2—CH2—CH2—NH—), 30.86 (O—CH2—CH2—CH2—NH—), 29.46 (C(CH3)3), 29.14 (C(CH3)3); HRMS (MALDI) was calculated to be 647.3035 for C30H43N6O10 (M+H+), and found to be 647.3031.
N4-Benzoyl-2′-O—(N,N′-di-boc-guanidinopropyl)-cytidine (2h) (516 mg, 798 μmol) was dissolved in dry pyridine (20 mL) and the solution was cooled in an ice bath. 4,4′-Dimethoxytrityl chloride (515 mg, 1.52 mmol) was added and the mixture was stirred overnight while the bath came up to room temperature. The reaction was quenched with methanol (10 mL) and the solvents were evaporated. The residue was purified by column chromatography using dichloromethane/methanol (99:1-98:2, v/v). The column was packed with solvent containing 1% triethylamine to yield 715 mg (94%) of the product as a pale yellow foam. 1H NMR (400 MHz, DMSO-d6) δ [ppm] 11.50 (s, 1H, NH), 11.29 (bs, 1H, NH), 8.43-8.37 (m, 2H, H6, NH—CH2—), 8.02-7.99 (m, 2H, benzoyl), 7.65-7.60 (m, 1H, benzoyl), 7.54-7.50 (m, 2H, benzoyl), 7.43-7.25 (m, 9H, DMTr), 7.18-7.15 (m, 1H, H5), 6.94-6.91 (m, 4H, DMTr), 5.88 (s, 1H, H1′), 5.04 (d, 1H, J=7.3 Hz, 3′-OH), 4.34-4.28 (m, 1H, H3′), 4.13-4.10 (m, 1H, H4′), 3.94-3.87 (m, 2H, H2′, 1×O—CH2—CH2—CH2—NH—), 3.76 (s, 6H, 2×OCH3), 3.76-3.70 (m, 1H, 1×O—CH2—CH2—CH2—NH—), 3.46-3.36 (m, 4H, 2×H5′, O—CH2—CH2—CH2—NH—), 1.86-1.80 (m, 2H, O—CH2—CH2—CH2—NH—), 1.42 (m, 9H, C(CH3)3), 1.36 (m, 9H, C(CH3)3); 13C NMR (75 MHz, DMSO-d6) δ [ppm] 167.19, 163.02, 158.11, 158.08, 155.12, 154.07, 151.93, 144.24 (C6), 135.47, 135.11, 133.06, 132.62, 129.70, 129.55, 128.35, 127.85, 127.73, 126.78, 113.19, 95.93 (C5), 88.99 (C1′), 85.90, 82.67 (C(CH3)3), 81.93 (C2′), 81.44 (C4′), 77.94 (C(CH3)3), 68.44 (O—CH2—CH2—CH2—NH—), 67.62 (C3′), 61.36 (C5′), 54.91 (OCH3), 54.90 (OCH3), 38.17 (O—CH2—CH2—CH2—NH—), 28.59 (O—CH2—CH2—CH2—NH—), 27.87 (C(CH3)3), 27.48 (C(CH3)3); HRMS (MALDI) was calculated to be 971.4161 for C51H60N6O12Na (M+Na+), and found to be 971.4181.
N4-Benzoyl-2′-O—(N,N′-di-boc-guanidinopropyl)-5′-O-(4,4′-dimethoxytrityl)-cytidine (2i) (683 mg, 720 μmol) was dissolved in dichloromethane (15 mL). 2-cyanoethyl N,N,N′,N′-tetraisopropylamino phosphane (274 μL, 864 μmol) and 4,5-dicyanoimidazole (98 mg, 828 μmol) were added. After stirring at room temperature for 5 h, TLC revealed that some starting material had not reacted. Therefore 10 mg of 4,5-dicyanoimidazole and 30 μL of the phosphitylation agent were added and the reaction was stirred at room temperature overnight. The solution was diluted with dichloromethane and washed with saturated sodium bicarbonate solution. After drying the organic layer over MgSO4 the solvent was evaporated and the residue was dissolved in a small amount (5 mL) of dichloromethane. This solution was dripped into a flask with hexane (500 mL) to form a white precipitate. Two thirds of the solvent was evaporated and the residual solvent was decanted carefully. The precipitate was redissolved in benzene and lyophilised to give 738 mg (89%) of 2d. According to 31P NMR spectrum the product was still containing a small amount of the hydrolysed phosphitylation reagent but this did not interfere with the oligonucleotide synthesis. 1H NMR (400 MHz, DMSO-d6) δ [ppm] 11.50-11.48 (m, 1H, NH), 11.25 (bs, 1H, NH), 8.52-8.45 (m, 1H, H6), 8.39-8.34 (m, 1H, NH—CH2—), 8.01-7.98 (m, 2H, benzoyl), 7.66-7.61 (m, 1H, benzoyl), 7.53-7.49 (m, 2H, benzoyl), 7.45-7.25 (m, 9H, DMTr), 7.13-7.09 (m, 1H, H5), 6.93-6.89 (m, 4H, DMTr), 5.95-5.92 (m, 1H, H1′), 4.56-4.38 (m, 1H, H3′), 4.31-4.28 (m, 1H, H4′), 4.07-3.29 (m, 17H), 2.90-2.57 (m, 2H, cyanoethyl), 1.86-1.78 (m, 2H, O—CH2—CH2—NH—), 1.40-1.35 (m, 18H, 2×C(CH3)3), 1.20-0.93 (m, 12H, iPr-CH3); 31P NMR (162 MHz, DMSO-d6) δ [ppm] 148.4, 148.0 (The signal of the hydrolised phosphitylation reagent appears at 13.9 ppm); HRMS (MALDI) was calculated to be 1149.5421 for C60H78N8O13P (M+H+), was found to be 1149.5447.
N3-Benzoyl-3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-uridine (3a) was synthesised according to a previously described procedure [22].
Compound 3a (1.14 g, 1.93 mmol) was dissolved in 9.6 mL of tert-butanol. Freshly distilled acrylonitrile (2.5 mL, 38.6 mmol) was added. After addition of cesium carbonate (645 mg, 1.98 mmol) the reaction was stirred for 4 h at room temperature. The reaction solution was filtered over celite. The residue was washed with 100 mL of dichloromethane. The filtrate was evaporated in vacuum. Purification via column chromatography in dichloromethane/ethyl acetate (99:1-95:5, v/v) yielded 746 mg (60%) of the desired product as a white powder. 1H NMR (250 MHz, acetone-d6) δ [ppm] 8.03-7.99 (m, 3H, H6, benzoyl), 7.79-7.72 (m, 1H, benzoyl), 7.61-7.55 (m, 2H, benzoyl), 5.80-5.74 (m, 2H, H5, H1′), 4.50-3.94 (m, 7H), 2.80-2.75 (m, 2H, O—CH2—CH2—CN), 1.17-1.07 (m, 28H, tetraisopropyl-CH and —CH3); 13C NMR (63 MHz, acetone-d6) δ [ppm] 171.11, 163.78, 151.06, 141.48, 136.94, 133.92, 132.20, 131.13, 119.80, 102.75, 91.47, 84.05, 83.69, 70.65, 68.08, 61.60, 20.35, 18.92, 18.91, 18.75, 18.73, 18.64, 18.50, 18.46, 18.40, 15.23, 14.79, 14.72, 14.43; HRMS was calculated to be 666.2637 for C31H45N3O8Si2Na (M+Na+) and found to be 666.2647.
Compound 3b (500 mg, 0.78 mmol) was dissolved in 10 mL of methanol in a glass tube suitable for the applied autoclave. Approximately 0.5 mL of the Raney-nickel slurry was put on a glass filter, washed thoroughly with dry methanol and rinsed into the glass tube with the solution of 3b. After addition of 5 mL methanol saturated with ammonia, the mixture was stirred for 1 h at room temperature in an autoclave under a hydrogen atmosphere (30 bar). The reaction solution was decanted from the catalyst into a glass filter. The catalyst was washed several times with methanol and the solvent was removed from the combined filtrates under reduced pressure. The product was purified on a silica gel column initially using dichloromethane/ethyl acetate (7:3-0:1, v/v) and thereafter ethyl acetate/methanol/triethylamine (6:3.5:0.5, v/v/v) to obtain 253 mg (60%) of a white powder. When we repeated the reduction we used the crude product after filtration and evaporation for further derivatisation. 1H NMR (250 MHz, acetone-d6) δ [ppm] 7.81 (d, 1H, J=8.1 Hz, H6), 5.71 (s, 1H, H1′), 5.53 (d, 1H, J=8.1 Hz, H5), 4.39-4.34 (m, 1H, H3′), 4.28-4.23 (m, 1H, H5′), 4.14-4.03 (m, 3H, H2′, H4′, H5′), 3.97-3.81 (m, 2H, O—CH2—CH2—CH2—NH2), 3.37-3.25 (m, 2H, O—CH2—CH2—CH2—NH), 1.92-1.82 (m, 2H, O—CH2—CH2—CH2—NH2), 1.14-1.05 (m, 28H, tetraisopropyl-CH and —CH3); 13C NMR (63 MHz, acetone-d6) δ [ppm] 167.18, 164.59, 151.93, 141.033, 102.82, 91.15, 83.77, 83.50, 70.88, 70.85, 61.78, 49.36, 33.03, 18.91, 18.90, 18.74, 18.61, 18.49, 18.47, 18.40, 15.19, 14.82, 14.71, 14.41; HRMS (MALDI) was calculated to be 544.2869 for C24H46N3O7Si2 (M+H+), and found to be 544.2880.
N,N′-Di-boc-N″-triflyl guanidine (320 mg, 0.82 mmol) was dissolved in 3.6 mL dichloromethane and triethylamine (150 μL) was added. The solution was cooled in an ice bath and 2′-O-(Aminopropyl)-3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)-uridine (3e) (490 mg, 0.9 mmol) was added. After 15 min the reaction mixture was removed from the ice bath was and stirred for 2.5 h at room temperature. The reaction solution was washed with saturated sodium bicarbonate solution and brine. After drying over Na2SO4 the solvent was evaporated in vacuum. The crude product was purified using column chromatography with dichloromethane/methanol (96:4-94:6, v/v). 410 mg (58%) of compound 3c were obtained. 1H NMR (400 MHz, DMSO-d6) δ [ppm] 11.49 (s, 1H, NH), 11.37 (m, 1H, NHuridine), 8.40-8.37 (m, 1H, NH—CH2—), 7.64 (d, 1H, J=7.9 Hz, H6), 5.64 (s, 1H, H1′), 5.53 (d, 1H, J=7.9 Hz, H5), 4.25-4.22 (H3′), 4.13-4.09 (m, 1H, H5′), 4.06-4.05 (m, 1H, H2′), 4.03-4.00 (m, 1H, H4′), 3.93-3.89 (m, 1H, H5′), 3.84-3.70 (m, 2H, O—CH2—CH2—CH2—CH2—NH—), 3.49-3.32 (m, 2H, O—CH2—CH2—CH2—NH—), 1.83-1.77 (m, 2H, O—CH2—CH2—CH2—NH—), 1.45 (s, 9H, C(CH3)3), 1.38 (s, 9H, C(CH3)3), 1.06-0.97 (m, 28H, tetraisopropyl-CH and —CH3); HRMS (MALDI) was calculated to be 808.3955 for C36H63N6O11Si2Na (M+Na+), and found to be 808.3991.
To a solution of compound 3c (910 mg, 1.16 mmol) and triethylamine (240 μL) in 13 mL tetrahydrofurane NEt3.3HF (700 μL, 4.3 mmol) was added. The reaction mixture was stirred for 1 h at room temperature. The solvents were evaporated and the residue was purified on a silica gel column using dichloromethane/methanol (93:7-92:8, v/v) to give 629 mg (97%) of a white foam. 1H NMR (250 MHz, acetone-d6) δ [ppm] 11.67 (bs, 1H, NH), 10.03 (bs, 1H, NH), 8.46-8.41 (m, 1H, NH—CH2—), 8.10 (d, 1H, J=8.2 Hz, H6), 5.99-5.97 (m, 1H, H1′), 5.58 (d, 1H, J=8.2 Hz, H5), 4.39-3.46 (m, 11H), 1.95-1.85 (m, 2H, O—CH2—CH2—CH2—NH—), 1.51 (s, 9H, C(CH3)3), 1.43 (s, 9H, C(CH3)3); 13C NMR (63 MHz, acetone-d6) δ [ppm] 165.64, 164.59, 157.88, 154.86, 152.37, 142.33, 103.31, 89.82, 86.67, 84.77, 84.74, 84.39, 79.91, 70.73, 70.69, 62.45, 40.20, 30.96, 29.51, 29.19; MS (ESI) was calculated to be 566.2 for C23H37N5O10Na (M+Na+), and found to be 567.0.
2′-O—(N,N′-Di-boc-guanidinopropyl)-uridine (3f) (588 mg, 1.08 mmol) was dissolved in 11.4 mL of dry pyridine and 4,4′-Dimethoxytrityl chloride (460 mg, 1.36 mmol) was added. The reaction solution was stirred at room temperature for 5 h. The reaction mixture was quenched with water and the solvents were evaporated. The residue was dissolved in dichloromethane, washed twice with saturated sodium bicarbonate solution (2×50 mL) and then twice with brine (2×50 mL). The organic layer was dried over Na2SO4 and the solvent was removed under reduced pressure. After purification using column chromatography with dichloromethane/methanol (97:3, v/v) containing 0.5% triethylamine, 785 mg (86%) of a yellow powder was obtained. The yellow impurity could not be separated on the column. 1H NMR (250 MHz, DMSO-d6) δ [ppm] 11.49 (s, 1H, NH), 11.37 (m, 1H, NH), 8.41-8.36 (m, 1H, NH—CH2—), 7.75 (d, 1H, J=8.1 Hz, H6), 7.40-7.23 (m, 9H, DMTr), 6.92-6.88 (m, 4H, DMTr), 5.83-5.82 (m, 1H, H1′), 5.29-5.25 (m, 1H, H5), 5.09-5.06 (m, 1H, 3′-OH), 4.23-3.88 (m, 3H), 3.74 (s, 6H, 2×O—CH3), 3.68-3.63 (m, 2H), 3.43-3.20 (m, 4H), 1.82-1.72 (m, 2H, O—CH2—CH2—CH2—NH—), 1.44 (s, 9H, C(CH3)3), 1.37 (s, 9H, C(CH3)3); HRMS (MALDI) was calculated to be 846.3920 for C44H56N5O12 (M+H+), and found to be 846.3946.
2′-O—(N,N′-Di-boc-guanidinopropyl)-5′-O-(4,4′-dimethoxytrityl)-uridine (3g) (770 mg, 0.9 mmol) was dissolved in dichloromethane (11 mL). To this solution, 2-cyanoethyl N,N,N′,N′-tetraisopropylamino phosphane (400 μL, 1.26 mmol) and 4,5-dicyanoimidazole (130 mg, 1.1 mmol) were added. The reaction progress was observed with TLC (dichloromethane/ethyl acetate 1:1 (v:v), containing 0.5% triethylamine). Because the reaction was not complete after two hours, an additional 0.3 equivalents of the reagents were added and the reaction was completed after additional 40 minutes. The resulting solution was washed twice with saturated sodium bicarbonate solution (2×100 mL) and once with brine (200 mL). After drying over Na2SO4 the solvent was evaporated and the residue was purified on a silica gel column with dichloromethane/ethyl acetate (6:4-1:1, v/v) containing 0.5% triethylamine. The mixture of the two diastereomers was obtained as a light yellow foam (762 mg, 83%). 1H NMR (400 MHz, DMSO-d6) δ [ppm] 11.50-11.48 (m, 1H, NH), 11.35 (bs, 1H, NH), 8.39-8.33 (m, 1H, NH—CH2—), 7.87-7.80 (m, 1H, H6), 7.41-7.22 (m, 9H, DMTr), 6.91-6.86 (m, 4H, DMTr), 5.86-5.84 (m, 1H, H1′), 5.23-5.18 (m, 1H, H5), 4.46-4.32 (m, 1H), 4.21-4.16 (m, 1H), 4.09-4.03 (m, 1H), 3.83-3.26 (m, 16H), 2.80-2.59 (m, 2H, —O—CH2—CH2—CN), 1.81-1.74 (m, 2H, O—CH2—CH2—CH2—NH—), 1.42-1.36 (m, 18H, C(CH3)3), 1.13-0.94 (m, 12H, iPr-CH3); 31H NMR (121 MHz, DMSO-d6) δ [ppm] 150.0, 148.6; HRMS (MALDI) was calculated to be 1046,4999 for C63H73N7O13P (M+H+), and found to be 1046,5021.
O6-(2,4,6-Triisopropylbenzenesulfonyl)-3′,5′-O-di-tert-butylsilanediyl guanosine (4a) was synthesised according to a previously described procedure [21].
Compound 4a (2.28 g, 3.3 mmol) was dissolved in tert-butanol (17 mL). Freshly distilled acrylonitrile (4.25 mL, 66 mmol) and cesium carbonate (1.16 g, 3.3 mmol) were added to the solution. After vigorous stirring at room temperature for 2-3 h, the mixture was filtered through celite. The solvent and excess reagents were removed in vacuum. The crude material was used for the next reaction without further purification. The residue was dissolved in 4 mL of a mixture of formic acid/dioxane/water (70:24:6, v/v/v). After stirring at room temperature for 1 h, water (150 mL) was added to the mixture and the solution extracted with dichloromethane. The organic layer was dried over Na2SO4 and the solvent was evaporated. The residue was purified using column chromatography with dichloromethane/methanol (9:1, v/v) to give 1.1 g (70% over 2 steps) of 4b as a colourless foam. 1H NMR (250 MHz, DMSO-d6) δ [ppm] 10.71 (bs, 1H, NH), 7.89 (s, 1H, H8), 6.45 (bs, 2H, NH2), 5.81 (s, 1H, H1′), 4.45-4.33 (m, 3H), 4.05-3.81 (m, 4H), 2.83-2.76 (m, 2H, O—CH2—CH2—CN), 1.06 (s, 9H, C(CH3)3), 1.01 (s, 9H, C(CH3)3); 13C NMR (63 MHz, DMSO-d6) δ [ppm] 156.51, 153.69, 150.50, 135.36, 118.71, 116.53, 87.25, 80.31, 76.35, 73.80, 66.64, 65.14, 27.12, 26.80, 22.07, 19.82, 18.29; MS (ESI) was calculated to be 477.2 for C21H33N6O5Si (M+H+), and found to be 477.5.
Compound 4b (500 mg, 1.06 mmol) was dissolved in dry methanol (5 mL). Raney nickel (ca. 0.5 mL of the methanol-washed sediment) and methanol (5 mL) saturated with ammonia were then added. The mixture was hydrogenated at 30 bar hydrogen-pressure for 1 h at room temperature. Thereafter the mixture was filtered through a glass filter and the catalyst was washed several times with methanol and a methanol/water mixture. The solvents were evaporated from the filtrate and the residue was used without further purification for the next reaction. MS (ESI) was calculated to be 481.3 for C21H37N6O5Si (M+H+), and found to be 481.8.
N,N′-Di-boc-N″-triflyl guanidine (163 mg, 0.415 mmol) was dissolved in dichloromethane (2.1 mL) and triethylamine (54 μL) was then added. The solution was cooled in an ice bath and then 2′-O-(2-Aminopropyl)-3′,5′-O-di-tert-butylsilanediyl guanosine (4e) (200 mg, 0.42 mmol) was added. After 30 minutes the reaction mixture was removed from the ice bath then stirred for an additional 30 minutes at room temperature. The reaction solution was washed with saturated sodium bicarbonate solution and brine. After drying over Na2SO4 the solvent was evaporated. The residue was purified by column chromatography using dichloromethane/methanol (9:1, v/v) to give 270 mg (89%) of 4c. 1H NMR (400 MHz, DMSO-d6) δ [ppm] 11.49 (bs, 1H, NH), 10.66 (bs, 1H, NH), 8.56-8.53 (m, 1H, NH—CH2—), 7.87 (s, 1H, H8), 6.39 (bs, 2H, NH2), 5.86 (s, 1H, H1′), 4.42-4.38 (m, 1H, H3′), 4.30-4.27 (m, 2H, H2′, H5′), 4.06-3.93 (m, 3H, H4′, H5′, ½×O—CH2—CH2—CH2—NH—), 3.72-3.67 (m, 1H, ½×O—CH2—CH2—CH2—NH—), 3.51-3.30 (m, 2H, O—CH2—CH2—CH2—NH—), 1.84-1.77 (m, 2H, O—CH2—CH2—CH2—NH—), 1.46 (s, 9H, —CO—C(CH3)3), 1.39 (s, 9H, —CO—C(CH3)3), 1.06 (s, 9H, —Si—C(CH3)3), 0.97 (s, 9H, —Si—C(CH3)3); HRMS (MALDI) was calculated to be 723.3856 for C32H55N5O9Si (M+H+), and found to be 723.3880.
A solution of compound 4c (400 mg, 0.55 mmol) in 3.6 mL of pyridine was cooled in an ice bath and isobutyryl chloride (145 μL, 1.37 mmol) was then added dropwise. The mixture was stirred at 0° C. for 1 h, then at room temperature for 1 h and evaporated to dryness. The residue was dissolved in 40 mL dichloromethane and washed twice with saturated sodium bicarbonate solution (2×60 mL) and once with brine (60 mL). The organic phase was dried over Na2SO4 and the solvent was evaporated. The residue was purified by column chromatography using dichloromethane/methanol (95:5-90:10, v/v) to give 318 mg (ca. 70%) of a mixture of mono- and di-isobutyryl derivative. 1H NMR (250 MHz, DMSO-d6) δ [ppm] 12.12 (s, 1H, NH), 11.57-11.51 (m, NH, NH-boc), 10.53 (s, NH-boc*), 8.54-8.49 (m, 2′-O—CH2—CH2—CH2—NH—), 8.25-8.22 (m, 1H, H8), 5.90-5.88 (m, 1H, H1′), 4.42-3.42 (m, 9H), 2.85-2.72 (m, 1.5H, —CH(CH3)2), 1.99-1.73 (m, 2H, 2′-O—CH2—CH2—CH2—NH—), 1.47-1.33 (m, 18H, 2×-CO—C(CH3)3), 1.15-0.99 (m, 27H, 2×—Si—C(CH3)3, —CH(CH3)2, —CH(CH3)2*). As a result of the mixture comprising mono- and diisobutyryl derivatives, some of the integrals could not be given as whole numbers. Thus, signals that depend only on the diisobutyryl compound are marked with an asterisk. MS (ESI) was calculated to be 793.4 for C36HeiN8O10Si (M+H+), and found to be 794.6.
A mixture of N2-Isobutyryl-2′-O—(N,N′-di-boc-guanidinopropyl)-3′,5′-O-di-tert-butylsilanediyl guanosine (4f) and N2-Isobutyryl-2′-O—(N,N′-di-boc-N″-isobutyryl-guanidinopropyl)-3′,5′-O-di-tert-butylsilanediyl guanosine (4f*) (490 mg, ca. 592 μmol) was dissolved in dry tetrahydrofurane (7 mL). Triethylamine (165 μL, 1.11 mmol) and Et3N-3HF (352 μL, 2.16 mmol) were then added. After stirring at room temperature for 1 h the solvent was evaporated. The residue was purified using column chromatography with dichloromethane/methanol (9:1, v/v) to give 322 mg (ca. 79%) of a mixture of N2-Isobutyryl-2′-O(N,N′-di-boc-guanidinopropyl)-guanosine and N2-Isobutyryl-2′-O—(N,N′-di-boc-N″-isobutyryl-guanidinopropyl)-guanosine as white foam. A small sample of the mixture was separated for NMR spectroscopy. NMR data is given for the mono-isobutyryl compound. 1H NMR (400 MHz, DMSO-d6) δ [ppm] 12.08 (s, 1H, NH), 11.65 (s, 1H, NH), 11.46 (s, 1H, NH), 8.29 (s, 1H, H8), 8.28-8.25 (m, 1H, NH—CH2—), 5.91 (d, 1H, J=6.0 Hz, H1′), 5.16 (d, 1H, J=4.8 Hz, 3′-OH), 5.06-5.03 (m, 1H, 5′-OH), 4.36-4.29 (m, 2H, H2′, H3′), 3.95-3.93 (m, 1H, H4′), 3.67-3.46 (m, 4H, 2×H5′, O—CH2—CH2—CH2—NH—), 3.33-3.28 (m, 2H, O—CH2—CH2—CH2—NH—), 2.77 (sep, 1H, J=6.8 Hz, —CH(CH3)2), 1.75-1.67 (m, 2H, O—CH2—CH2—CH2—NH—), 1.45 (s, 9H, —CO—C(CH3)3), 1.37 (s, 9H, —CO—C(CH3)3), 1.12 (d, 6H, J=6.8 Hz, —CH(CH3)2); 13C NMR (63 MHz, CDCl3) δ [ppm] 178.72, 163.52, 156.12, 155.16, 153.39, 147.73, 147.05, 138.81, 122.49, 88.47, 86.74, 83.65, 82.28, 79.58, 70.69, 69.87, 62.66, 38.87, 36.39, 29.32, 28.28, 28.11, 18.96, 18.89; HRMS (MALDI) was calculated to be 653.3253 for C28H45N8O10 (M+H+), and found to be 653.3274.
A mixture of N2-Isobutyryl-2′-O—(N,N′-di-boc-guanidinopropyl)-guanosine (4g) and N2-Isobutyryl-2′-O—(N,N′-di-boc-N″-isobutyryl-guanidinopropyl)-guanosine (4g*) (400 mg, ca. 583 μmol) was dissolved in dry pyridine (11 mL). 4,4′-Dimethoxytrityl chloride (280 mg, 0.82 mmol) was added and the solution was stirred for 3 h at room temperature. TLC revealed that some starting material remained at this time and an additional 0.3 equivalents of 4,4′-Dimethoxytrityl chloride were therefore added. When TLC demonstrated that the starting material had been consumed, the reaction was quenched with water and the solvents evaporated. The residue was purified by column chromatography using dichloromethane/methanol (98:2, v/v) containing 0.5% triethylamine to give 427 mg (ca. 74%) of the desired products. 1H NMR (400 MHz, DMSO-d6) δ [ppm] 12.09 (s, 1H, NH), 11.58 (s, 1H, NH), 11.47 (s, 0.5H, NH-boc), 10.50 (s, 0.5H, NH-boc*), 8.33-8.30 (m, 0.5H, 2′-O—CH2—CH2—CH2—NH—), 8.15-8.12 (m, 1H, H8), 7.35-7.18 (m, 9H, DMTr), 6.84-6.80 (m, 4H, DMTr), 5.97-5.94 (m, 1H, H1′), 5.15-5.13 (m, 1H, 3′-OH), 4.42-4.37 (m, 1H, H2′), 4.35-4.30 (m, 1H, H3′), 4.09-4.03 (m, 1H, H4′), 3.72 (s, 6H, 2×O—CH3), 3.69-3.47 (m, 2H, 2′-O—CH2—CH2—CH2—NH—), 3.37-3.26 (m, 3H, 2′-O—CH2—CH2—CH2—NH—, H5′), 3.17-3.13 (m, 1H, H5′), 2.79-2.73 (m, 1.5H, —CH(CH3)2), 1.77-1.67 (m, 2H, 2′-O—CH2—CH2—CH2—NH—), 1.43-1.35 (m, 18H, 2×-CO—C(CH3)3), 1.13-1.10 (m, 6H, —CH(CH3)2), 1.00-0.98 (m, 3H, —CH(CH3)2*). As a result of the mixture comprising mono- and diisobutyryl derivatives, some of the integrals could not be given as whole numbers. Thus, signals that depend only on the diisobutyryl compound are marked with an asterisk. MS (ESI) was calculated to be 955.5 for C49H63N8O12 (M+H+), and found to be 956.5.
A mixture of N2-Isobutyryl-2′-O—(N,N′-di-boc-guanidinopropyl)-5′-O-(4,4′-dimethoxy trityl)-guanosine (4h) and N2-Isobutyryl-2′-O—(N,N′-di-boc-N″-isobutyryl-guanidinopropyl)-5′-O-(4,4′-dimethoxytrityl)-guanosine (4h*) (380 mg, ca. 384 μmol) was dissolved in dichloro methane (8 mL), then 2-cyanoethyl N,N,N′,N′-tetraisopropylamino phosphane (160 μL, 0.52 mmol) and 4,5-dicyanoimidazole (57 mg, 0.5 mmol) were added. After 2 h TLC showed complete consumption of the starting material. The reaction solution was then washed twice with saturated sodium bicarbonate solution (2×50 mL) and once with brine (100 mL). After drying over Na2SO4 the solvent was evaporated and the residue was purified using column chromatography with dichloromethane/ethyl acetate (8:2, v/v) containing 0.5% triethylamine to give 350 mg (ca. 76%) of the two diastereomers of 4d and 4d*. 1H NMR (400 MHz, DMSO-d6) δ [ppm] 12.11 (bs, 1H, NH), 11.61-11.57 (m, 1H, NH), 11.46-11.44 (m, 0.5H, NH-boc), 10.50-10.46 (m, 0.5H, NH-boc*), 8.31-8.27 (m, 0.5H, 2′-O—CH2—CH2—CH2—NH—), 8.18-8.14 (m, 1H, H8), 7.36-7.19 (m, 9H, DMTr), 6.85-6.80 (m, 4H, DMTr), 5.97-5.88 (m, 1H, H1′), 4.64-4.61 (m, 1H, H2′), 4.44-4.37 (m, 1H, H3′), 4.27-4.12 (m, 1H, H4′), 3.79-3.18 (m, 10H), 3.72 (s, 6H, 2×OCH3), 2.81-2.70 (m, 2.5H, —CH(CH3)2), 2.60-2.47 (m, 2H, —P—O—CH2—CH2—CN), 1.75-1.65 (m, 2H, 2′-O—CH2—CH2—CH2—NH—), 1.41-1.34 (m, 18H, 2×-CO—C(CH3)3), 1.15-1.10 (m, 18H, —N((CH(CH3)2)2, —CO—CH(CH3)2), 1.00-0.96 (m, 3H, —CH(CH3)2*); 31P NMR (162 MHz, DMSO-d6) δ [ppm] 149.59, 149.44, 149.52, 149.19. As a result of the mixture comprising mono- and diisobutyryl derivatives, some of the integrals could not be given as whole numbers. Thus, signals that depend only on the diisobutyryl compound are marked with an asterisk. MS (ESI) was calculated to be 1155.6 for C68H60N10O13P (M+H+), and found to be 1157.3.
To circumvent the problem of a product mixture upon introduction of the isobutyryl group to the N2-position of guanosine, we established a different protection strategy, using the dimethylformamidine group (
Compound 4c (1.12 g, 1.55 mmol) was dissolved in 25 mL dry methanol. N,N-Dimethylformamide dimethyl acetal (1.0 mL, 7.76 mmol) was added and the solution was stirred at room temperature overnight. After a reaction time of 12 h the solvents were removed in vacuum and the residue was purified by column chromatography using dichloromethane/methanol (19:1, v/v) to give 1.14 g (94%) of the dmf-protected derivative. 1H NMR (400 MHz, DMSO-d6) δ [ppm] 11.51 (s, 1H, N1H), 11.40 (s, 1H, NH-boc), 8.54 (s, 1H, —N═CH—N(CH3)2), 8.47 (m, 1H, 2′-O—CH2—CH2—CH2—NH—), 7.99 (s, 1H, H-8), 5.98 (s, 1H, H1′), 4.48-4.45 (m, 1H, H5′), 4.41-4.39 (m, 1H, H2′), 4.33-4.30 (m, 1H, H5′), 4.07-3.99 (m, 2H, H3′ und H4′), 3.98-3.77 (m, 2H, 2′-O—CH2—CH2—CH2—NH—), 3.48-3.37 (m, 2H, 2′-O—CH2—CH2—CH2—NH—), 3.14 (s, 3H, N—CH3), 3.04 (s, 3H, N—CH3), 1.87-1.78 (m, 2H, 2′-O—CH2—CH2—CH2—NH—), 1.47 (s, 9H, —CO—C(CH3)3), 1.37 (s, 9H, —CO—C(CH3)3), 1.06 (s, 9H, —Si—C(CH3)3), 1.00 ppm (s, 9H, —Si—C(CH3)3); 13C NMR (75 MHz, DMSO-d6) δ [ppm] 163.00, 157.60, 157.39, 157.35, 154.98, 151.95, 149.21, 136.96, 119.86, 88.07, 82.78, 80.59, 77.90, 76.48, 73.83, 69.64, 66.77, 44.41, 40.58, 34.54, 28.61, 27.86, 27.44, 27.08, 26.70, 22.06, 19.76; HRMS (MALDI) was calculated to be 800.4097 for C35H59N9O9SiNa (M+Na+), and found to be 800.4124.
N2-Dimethylformamidine-2′-O—(N,N′-di-boc-guanidinopropyl)-3′,5′-O-di-tert-butylsilanediyl guanosine (1.24 g, 1.59 mmol) was dissolved in dry tetrahydrofurane (17 mL). Triethylamine (470 μL, 3.18 mmol) and Et3N.3HF (943 μL, 5.79 mmol) were then added. After stirring at room temperature for 1 h the solvent was evaporated. The residue was purified using column chromatography with dichloromethane/methanol (9:1, v/v) to give 840 mg (83%) of N2-Dimethylformamidine-2′-O—(N,N′-di-boc-guanidinopropyl)-guanosine as white foam. 1H NMR (400 MHz, DMSO-d6) δ [ppm] 11.50 (s, 1H, N1H), 11.34 (s, 1H, NH-boc), 8.54 (s, 1H, —N═CH—N(CH3)2), 8.35 (m, 1H, 2′-O—CH2—CH2—CH2—NH—), 8.10 (s, 1H, H8), 5.95-5.94 (m, 1H, H1′), 5.14-5.12 (m, 1H, 3′-OH), 5.08-5.05 (m, 1H, 5′-OH), 4.31-4.30 (m, 2H, H2′, H3′), 3.95-3.93 (m, 1H, H4′), 3.67-3.56 (m, 4H, 2×H5′, O—CH2—CH2—CH2—NH—), 3.36-3.33 (m, 2H, O—CH2—CH2—CH2—NH—), 3.16 (s, 3H, N—CH3), 3.04 (s, 3H, N—CH3), 1.77-1.74 (m, 2H, O—CH2—CH2—CH2—NH—), 1.47 (s, 9H, —CO—C(CH3)3), 1.37 (s, 9H, —CO—C(CH3)3); 13C NMR (75 MHz, DMSO-d6) δ [ppm] 162.98, 157.84, 157.44, 157.24, 155.10, 151.89, 149.61, 136.41, 119.77, 85.26, 85.23, 82.75, 81.36, 78.00, 68.51, 67.87, 60.81, 40.54, 37.86, 34.53, 28.65, 27.85, 27.50; HRMS (MALDI) was calculated to be 660.3076 for O27H43N9O9Na (M+Na+), and found to be 660.3087.
N2-Dimethylformamidine-2′-O—(N,N′-di-boc-guanidinopropyl)-guanosine (840 mg, 1.32 mmol) was dissolved in dry pyridine (30 mL). 4,4′-Dimethoxytrityl chloride (670 mg, 1.98 mmol) was added and the solution was stirred for 3 h at room temperature. After the reaction was complete according to TLC, the reaction was quenched with methanol and the solvents were evaporated. The residue was purified by column chromatography using dichloromethane/methanol (100:0->95:5, v/v; the column was packed with dichloromethane containing 0.5% triethylamine) to give 1.08 g (87%) of the desired product. 1H NMR (400 MHz, DMSO-d6) δ [ppm] 11.51 (s, 1H, N1H), 11.38 (s, 1H, NH-boc), 8.50 (s, 1H, —N═CH—N(CH3)2), 8.40 (m, 1H, 2′-O—CH2—CH2—CH2—NH—), 7.94 (s, 1H, H8), 7.38-7.20 (m, 9H, DMTr), 6.86-6.82 (m, 4H, DMTr), 6.01-6.00 (m, 1H, H1′), 5.16-5.13 (m, 1H, 3′-OH), 4.35-4.30 (m, 2H, H2′, H3′), 4.08-4.05 (m, 1H, H4′), 3.73 (s, 6H, 2×O—CH3), 3.71-3.61 (m, 2H, 2′-O—CH2—CH2—CH2—NH—), 3.40-3.35 (m, 2H, 2′-O—CH2—CH2—CH2—NH—), 3.28-3.16 (m, 2H, 2×H5′), 3.09 (s, 3H, N—CH3), 3.02 (s, 3H, N—CH3), 1.80-1.74 (m, 2H, 2′-O—CH2—CH2—CH2—NH—), 1.44 (s, 9H, —CO—C(CH3)3), 1.34 (s, 9H, —CO—C(CH3)3); 13C NMR (100 MHz, DMSO-d6) δ [ppm] 162.96, 157.97, 157.95, 157.72, 157.48, 157.20, 155.10, 151.89, 149.59, 144.71, 136.15, 135.43, 135.30, 129.59, 129.57, 127.67, 127.57, 126.55, 119.83, 113.02, 85.53, 85.37, 82.72, 82.69, 81.04, 77.96, 69.02, 68.22, 63.55, 54.90, 40.54, 37.99, 34.54, 28.66, 27.82, 27.49; HRMS (MALDI) was calculated to be 962.4383 for C48H61N9O11Na (M+Na+), and found to be 962.4408.
N2-Dimethylformamidine-2′-O—(N,N′-di-boc-guanidinopropyl)-5′-O-(4,4′-dimethoxytrityl)-guanosine (1.22 g, 1.3 mmol) was dissolved in dichloromethane (25 mL), then 2-cyanoethyl N,N,N′,N′-tetraisopropylamino phosphane (590 μL, 1.76 mmol) and 4,5-dicyanoimidazole (199 mg, 1.69 mmol) were added. After 4 h TLC showed complete consumption of the starting material. The reaction solution was then washed twice with saturated sodium bicarbonate solution and once with brine. After drying over Na2SO4 solvent was evaporated and the residue was purified using column chromatography with dichloromethane/acetone/methanol (4:0:1->3:0:2->2:1:2->2:2:1, v/v, the column was packed with eluent containing 0.5% triethylamine). The residue was dissolved in a small amount (5 mL) of dichloromethane. This solution was dripped into a flask with hexane (500 mL) to form a white precipitate. Two thirds of the solvent was evaporated and the residual solvent was decanted carefully. The precipitate was redissolved in benzene and lyophilised to give 1.01 mg (68%) of the phosphoramidite. According to 31P NMR spectrum the product was still containing a small amount of the hydrolysed phosphitylation reagent but this did not interfere with the oligonucleotide synthesis. 1H NMR (400 MHz, DMSO-d6) δ [ppm] 11.50-11.48 (m, 1H, NH), 11.38 (G, 1H, NH), 8.44-8.42 (m, 1H, —N═CH—N(CH3)2), 8.39-8.34 (m, 1H, 2′-O—CH2—CH2—CH2—NH—), 7.96 (s, 1H, H8), 7.37-7.19 (m, 9H, DMTr), 6.85-6.78 (m, 4H, DMTr), 6.07-6.05 (m, 1H, H1′), 4.64-4.58 (m, 1H, H3′), 4.48-4.44 (m, 1H, H2′), 4.26-4.19 (m, 1H, H4′), 3.80-3.23 (m, 10H), 3.73-3.70 (m, 6H, 2×OCH3), 3.07 (s, 3H, N—CH3), 3.02 (s, 3H, N—CH3), 2.77-2.74 (m, 1H, —P—O—CH2—CH2—CN), 2.55-2.52 (m, 1H, —P—O—CH2—CH2—CN), 1.80-1.72 (m, 2H, 2′-O—CH2—CH2—CH2—NH—), 1.44-1.34 (m, 18H, 2×-CO—C(CH3)3), 1.20-0.93 (m, 12H, —N((CH(CH3)2)2); 31P NMR (121 MHz, DMSO-d6) δ [ppm] 149.21, 148.93.
We also established an alternative reduction procedure according to a literature procedure [30].
This procedure was tested with 2′-O-(Cyanomethyl)-3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)uridine. 2′-O-(Cyanomethyl)-3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl)uridine and 1 equivalent of dried (waterfree) Ni(II)Cl2 was dissolved in absolute ethanol. 6 equivalents of sodium borohydride were added in small portions. After 4 h the reaction was quenched with 10 equivalents of diethylene triamine. The solvents were evaporated and the residue was dissolved in ethyl acetate. The solution was washed with saturated sodium bicarbonate solution and dried over MgSO4. After evaporation of the solvent the residue was purified by column chromatography using dichloromethane/methanol (5:1, v/v) to give 2′-O-Aminoethyl-3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl) uridine (ca. 30%).
The obtained phosphoramidites where used for synthesis of the GP-modified siRNA antisense strands depicted in Table 1 and 2, and for the synthesis of GP-modified siRNA sense strands depicted in Table 3.
Modified oligonucleotides were synthesised on an Expedite 8909 synthesiser using phosphoramidite chemistry. The 2′-O-guanidinopropyl-modified nucleosides were inserted into the HBV antisense strand (intended guide, 5′-UUG AAG UAU GCC UCA AGG UCG-3′) (SEQ ID NO: 1) at each of positions 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21 from the 5′ end (Table 1). In some antisense oligonucleotides, a combination of two (positions 2 & 5, 2 & 3, and 19 & 20), three (positions 2, 5 & 17 and 2, 3 & 17) or four (positions 2, 5, 17 & 20) 2′-O-guanidinopropyl-modifications was included (Table 2). The sense strand oligonucleotide 5′-ACC UUG AGG CAU ACU UCA AdTdT-3′ (SEQ ID NO: 2) included a single 2′-O-guanidinopropyl-modification at positions 17 or a combination of three 2′-O-guanidinopropyl-modification at positions 5, 13 and 17 (Table 3). The duplex HBV siRNA3 targeted HBV genotype A coordinates 1693 to 1711 (
Initially, to measure knockdown efficiency of 2′-O-guanidinopropyl-modified siRNAs in situ, HEK293 cells were co-transfected with RNAi activators together with a reporter gene plasmid (psiCHECK-HBx) [20] (
Huh7 and HEK293 cells were cultured in DMEM (Lonza, Basel, Switzerland) supplemented with 5% foetal calf serum (Gibco BRL, UK). Cells were seeded in 24-well plates at a confluency of 40% on the day before transfection, and were then maintained in antibiotic-free medium for at least an hour prior to transfection. To assess target knockdown when using the luciferase reporter assay, Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) was employed to transfect HEK293 cells with 100 ng psiCHECK-HBx [20] and 32.5 ng siRNA (5 nM final concentration) at ratios of 1:1 and 1:3 (ml:mg) respectively. The psiCHECK-HBx reporter vector contains the HBx target sequence downstream of the Renilla ORF within the psiCHECK 2.2 (Promega, Wis., USA) and has been described previously [20]. Forty-eight hours after transfection, cells were assayed for luciferase activity using the Dual-Luciferase® Reporter Assay System (Promega, Wis., USA) and the ratio of Renilla luciferase to Firefly luciferase activity was calculated. Similarly, to assess knockdown of HBV replication in a liver-derived line, Huh7 cells were transfected with 100 ng pCH-9/3091 [28] and 32.5 ng siRNA. Forty eight hours after transfection, growth medium was harvested and HBsAg concentration was measured by ELISA using the MONOLISA® HBs Ag ULTRA kit (Bio-Rad, CA, USA). Each experiment was repeated at least in triplicate.
Data have been expressed as the mean±standard error of the mean. Statistical difference was considered significant when P<0.05 and was determined according to the student's t-test and calculated with the GraphPad Prism software package (GraphPad Software Inc., CA, USA).
To assess efficacy against HBV replication in vitro, Huh7 liver-derived cells were co-transfected with siRNAs together with the pCH-9/3091 HBV replication competent target plasmid (
siRNAs containing 2′-O-guanidinopropyl (GP) modifications were incubated in the presence or absence of 80% fetal calf serum (FCS) for time intervals of 0 to 24 hours to assess their stability (
Cell culture, transfection and RNA extraction. HEK293 cells were cultured and transfected as described previously. Briefly, cells were maintained in DMEM supplemented with 10% FCS, penicillin (50 IU/ml) and streptomycin (50 μg/ml) (Gibco BRL, UK). On the day prior to transfection, 250 000 HEK293 cells were seeded in dishes of 2 cm diameter. Transfection was carried out with 800 ng of unmodified or GP-containing siRNA using Lipofectamine (Invitrogen, CA, USA) according to the manufacturer's instructions. As a positive control for the induction of the interferon (IFN) response, cells were also transfected with 800 ng poly (I:C) (Sigma, Mich., USA). Two days after transfection, RNA was extracted with Tri Reagent (Sigma, Mich., USA) according to the manufacturer's instructions.
Real Time Quantitative PCR of Interferon Response Genes.
To assess induction of the interferon (IFN) response genes, IFN-β and GAPDH cDNA preparation and amplification where performed according to the procedures described by Song et al [31]. All qPCRs were carried out using the Roche Lightcycler V.2. Controls included water blanks and RNA extracts that were not subjected to reverse transcription. Tag readymix with SYBR green (Sigma, Mo., USA) was used to amplify and detect DNA during the reaction. Thermal cycling parameters consisted of a hotstart for 30 sec at 95° C. followed by 50 cycles of 58° C. for 10 sec, 72° C. for 7 sec and then 95° C. for 5 sec. Specificity of the PCR products was verified by melting curve analysis and agarose gel electrophoresis. The primer combinations used to amplify IFN-β mRNA and GAPDH mRNA of human HEK293 cells are set out in Table 4.
Interferon Response Gene Induction in Transfected Cells.
The principle of the sensitive cell viability assay is based on conversion of the yellow 3-(4,5-dimethylhiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to a dark blue/purple product by mitochondrial succinyl dehydrogenase. The insoluble product is solubilised in a solvent (dimethylsyulphoxide, DMSO) and the concentration measured spectrophotometrically by determining the optical density ratio at 570 nm, which shows the concentration of MTT product, to that at 655 nm, which indicates the number of cells that was analysed in each assay. Since conversion of the substrate to product can only occur in metabolically active cells, the activity of the mitochondrial dehydrogenase can be used conveniently as a measure of cell viability. Procedures were followed according to the recommendations of the supplier of the MTT (Sigma, Mich., USA). MTT cells were plated in 125 μl media per well in a 96-well plate, then incubated overnight (at 37° C., 5% CO2) to allow cells to attach. Cells were then transfected with unmodified or GP-modified siRNAs (2.5 nM or 8.125 ng per well) and gently mixed by shaking. Cells were then cultured for a further 1-5 days. MTT substrate (Sigma, Mich., USA) was freshly prepared by dissolving in 1×Dulbecco's Phosphate Buffered Saline (DPBS) or Phosphate Buffered Saline (PBS) at a concentration of 5 mg/ml. Twenty μl of the MTT solution was added to each well and gently mixed for 5 minutes. Thereafter the plates were incubated for a further 1-5 hours to allow metabolism of MTT. The medium was then gently removed from each well. The blue MTT metabolic product, formazan, was resuspended in 200 μl DMSO and gently mixed by shaking for 5 minutes. The spectrophotometric optical density was measured at 570 nm and divided by the background reading at 655 nm.
A panel of dual luciferase reporter plasmids was generated in which the HBV target sequences included variable numbers of nucleotides that were complementary to the intended siRNA3 guide strand. The targets in the reporter plasmids are listed below:
The structures of the dual luciferase reporters are illustrated schematically in
The procedure for inserting CT, IT1, IT2 and SO into psiCHECK-HBx is summarised as follows, to generate the backbone for cloning of the inserts, psiCHECK-HBx (2 μg) was digested with XhoI and NotI to generate 6242 bp and 564 bp fragments. The 6242 bp psiCHECK fragment was purified from an agarose gel using a gel extraction kit (Qiagen MinElute Gel Extraction Kit), according to Manufacturer's instructions. Yield was checked after electrophoresis on a 1% agarose gel. To generate the panel of siRNA3 target sequences, the oligonucleotides listed in Table 5 below were synthesized by Integrated DNA Technologies (IDT, Iowa, USA). Twenty microliters from a 100 μM stock of each of forward and reverse oligo were combined then heated to 95° C. for 5 minutes. Thereafter, the solutions were allowed to cool to room temperature. The annealed oligonucleotides, which had sticky ends complementary to those generated by NotI and XhoI restriction digestion, were then diluted with water to a concentration of 10 mM.
The annealed oligonucleotides were then ligated to the digested and purified psiCHECK backbone fragment according to standard procedures. Colonies were screened by restriction digestion of isolated plasmids using PvuII. Positive clones were verified by sequencing (Inqaba Biotech, South Africa).
To measure knockdown efficiency of 2′-O-guanidinopropyl-modified siRNAs that were completely or partially complementary to targets, Huh7 cells were co-transfected with various unmodified or GP-containing siRNAs, together with a reporter gene plasmid (psiCHECK-CT, psiCHECK-IT1, psiCHECK-IT2, psiCHECK-SO) [20] (
Compared to a scrambled siRNA control, analysis showed that the Renilla luciferase activity was diminished by at least 85% when the reporter plasmid containing the complete target was co-transfected with the unmodified or GP-modified siRNA (
Hydrodynamic Injection of Mice.
The murine hydrodynamic tail vein injection (HDI) method was employed to determine the effects of unmodified and GP-modified siRNAs on the expression of HBV genes in vivo. Experiments on animals were carried out in accordance with protocols approved by the University of the Witwatersrand Animal Ethics Screening Committee. A saline solution comprising 10% of the mouse's body mass was injected via the tail vein over 5-10 seconds. This saline solution included a combination of three plasmid vectors: 15 μg target DNA (pCH-9/3091); 25 μg anti-HBV siRNAs (unmodified siRNA3, GP3, GP4 and GP5), control non-targeting scrambled siRNA or no siRNA (saline control); and 5 μg pCl neo EGFP (a control for hepatic DNA delivery, which constitutively expresses the EGFP marker gene [32]). Each experimental group comprised 5 mice. Blood was collected under anaesthesia by retroorbital puncture on days 3 and 5 after HDI. Serum HBsAg concentration was measured using the Monolisa (ELISA) immunoassay kit (BioRad, CA, USA) according to the manufacturer's instructions. To measure effects of siRNAs on circulating viral particle equivalents (VPEs), total DNA was isolated from 50 μl of the serum of mice on days 3 and 5 after hydrodynamic injection and viral DNA determined using quantitative PCR according to previously described methods [17]. Briefly, total DNA was isolated from 50 μl of mouse serum using the Total Nucleic Acid Isolation Kit and MagNApure instrument from Roche Diagnostics. Controls included water blanks and HBV negative serum. DNA extracted from the equivalent of 8 μl of mouse serum was amplified using SYBR green Taq readymix (Sigma, Mo., USA). Crossing point analysis was used to measure virion DNA concentrations and standard curves were generated using EuroHep calibrators [33]. The HBV surface primer set was: HBV surface forward: 5′-TGC ACC TGT ATT CCA TC-3′ (SEQ ID NO: 52), and HBV surface reverse: 5′-CTG AAA GCC AAA CAG TGG-3′ (SEQ ID NO: 53). PCR was carried out using the Roche Lightcycler V.2. Capillary reaction volume was 20 μl and thermal cycling parameters consisted of a hot start for 30 sec 95° C. followed by 50 cycles of 57° C. for 10 sec, 72° C. for 7 sec and then 95° C. for 5 sec. Specificity of the PCR products was verified by melting curve analysis and agarose gel electrophoresis.
Inhibition of Markers of HBV Replication In Vivo.
The influence of 2′-O-guanidinopropyl-modified nucleosides on thermal stability of different RNA duplexes was examined. For this purpose, the GGP and UGP modified phosphoramidites were inserted into 12mer RNA (ON2-ON6) and the duplex melting point was measured. As shown in Table 6, the presence of 2′-O-guanidinopropyl group in oligoribonucleotides did not significantly affect the stability of duplexes, although a slight trend to destabilisation was observed. Guanidinopropyl modified building blocks gives almost the same Tm value for single, double and triple substituted oligonucleotides.
Interestingly, in one case when a 2′-O-guanidinopropyl modification of G was placed in a central position, the Tm decreased more significantly (ΔTm=−2,4° C.).
The results indicate that the thermodynamic effect of 2′-O-guanidinopropyl group is independent on the placement of the modification and which of the nucleosides is modified. After including more, but not adjacent substitutions, an additional destabilising effect was not observed (ON5 and ON6, Table 6) Moreover, for the modified oligonucleotides bearing more than one 2′-O-guanidinopropyl residue, high binding affinity to the complementary strand, was unaffected.
This observation is in accordance with the hybridisation properties of oligonucleotides containing 2′-O-aminopropyl (2′-O-AP) groups [16]. Incorporation of single 2′-O-AP units at the 3′-end or in the middle of an oligomer reduce the Tm of an RNA duplex. When adjacent residues are modified or when all nucleotides of a strand are substituted with 2′-O-AP groups duplex stabilisation occurs. Molecular dynamic and NMR data confirmed that flexibility of the aminoalkyl chain did not result in formation of strong electrostatic interactions or hydrogen bond formation [16]. Moreover, no or little stabilising effect is expected to be associated with the degree of hydration for the 2′-O-AP [22], [37].
As a result of the flexibility of the 2′-O-guanidinopropyl residue, local disruption and thermodynamic destabilisation of the modified duplexes is expected. However, presence of the guanidinium group, with three planar nitrogen atoms, allows protonation over a wide pH range. This should neutralise overall negative charge and preserve thermodynamic stability of 2′-O-guanidinopropyl-modified RNA.
Transfections of Huh7 cells were carried out as has been described above. Briefly, 2′-O-guanidinopropyl-modified siRNAs comprising various sense and antisense combinations were used to co-transfect HEK293 cells together with a reporter gene plasmid (psiCHECK-HBx) [8] (
Efficacy against the HBV targets of siRNAs comprising strands that had single modifications in both the sense or antisense strands was similar to the unmodified siRNA3 (
| Number | Date | Country | Kind |
|---|---|---|---|
| 2011/07890 | Oct 2011 | ZA | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2012/055915 | 10/26/2012 | WO | 00 | 4/25/2014 |
