RNA RECOGNITION BY FLUOR-AROMATIC SUBSTITUTED

RNA exhibits a higher structural diversity than DNA and is an important molecule in the biology of life. It shows a number of secondary structures such as duplexes, hairpin loops, bulges, internal loops, etc. However, in natural RNA, bases are limited to the four predominant structures U, C, A, and G and so the number of compounds that can be used for investigation of parameters of base stacking, base pairing, and hydrogen bond is limited. We synthesized different fluoromodifications of RNA building blocks: 1′-deoxy-1′-phenyl-β-d-ribofuranose (B), 1′-deoxy-1′-(4-fluorophenyl)-β-d-ribofuranose (4 FB), 1′-deoxy-1′-(2,4-difluorophenyl)-β-d-ribofuranose (2,4 DFB), 1′-deoxy-1′-(2,4,5-trifluorophenyl)-β-d-ribofuranose (2,4,5 TFB), 1′-deoxy-1′-(2,4,6-trifluorophenyl)-β-d-ribofuranose, 1′-deoxy-1′-(pentafluorophenyl)-β-d-ribofuranose (PFB), 1′-deoxy-1′-(benzimidazol-1-yl)-β-d-ribofuranose (BI), 1′-deoxy-1′-(4-fluoro-1H-benzimidazol-1-yl)-β-d-ribofuranose (4 FBI), 1′-deoxy-1′-(6-fluoro-1H-benzimidazol-1-yl)-β-d-ribofuranose (6 FBI), 1′-deoxy-1′-(4,6-difluoro-1H-benzimidazol-1-yl)-β-d-ribofuranose (4,6 DFBI), 1′-deoxy-1′-(4-trifluoromethyl-1H-benzimidazol-1-yl)-β-d-ribofuranose (4 TFM), 1′-deoxy-1′-(5-trifluoromethyl-1H-benzimidazol-1-yl)-β-d-ribofuranose (5 TFM), and 1′-deoxy-1′-(6-trifluoromethyl-1H-benzimidazol-1-yl)-β-d-ribofuranose (6 TFM). These amidites were incorporated and tested in a defined A, U-rich RNA sequence (12-mer, 5′-CUU UUC XUU CUU-3′ paired with 3′-GAA AAG YAA GAA-5′). Only one position was modified, marked as X and Y, respectively. UV melting profiles of those oligonucleotides were measured.     

Synthesis of modified RNA-oligonucleotides for structural investigations

RNA exhibits a higher structural diversity than DNA and is an important molecule in biology of life. It shows a number of secondary structures such as duplexes, hairpin loops, bulges, internal loops etc. However, in natural RNA, bases are limited to the four predominant structures U, C, A, and G and so the number of compounds that can be used for investigation of parameters of base stacking, base pairing and hydrogen bond, is limited. We synthesized different fluoromodifications of RNA building blocks: 1'-deoxy-1'-(2,4,6-trifluorophenyl)-beta-D-ribofuranose (F), 1'-deoxy-1'-(2,4,5-trifluorophenyl)-beta-D-ribofuranose (M) and 1'-deoxy-1'-(5-trifluoromethyl-1H-benzimidazol-1-yl)-beta-D-ribofuranose (D). Those amidites were incorporated and tested in a defined A, U-rich RNA sequence (12-mer, 5'-CUU UUC XUU CUU-3' paired with 3'-GAA AAG YAA GAA-5') (Schweitzer, B.A.; Kool, E.T. Aromatic nonpolar nucleosides as hydrophobic isosters of pyrimidine and purine nucleosides. J. Org. Chem. 1994, 59, 7238 pp.). Only one position was modified, marked as X and Y respectively. UV melting profiles of those oligonucleotides were measured.     

Synthesis of Modified RNA-Oligonucleotides for Structural Investigations

Abstract

RNA exhibits a higher structural diversity than DNA and is an important molecule in biology of life. It shows a number of secondary structures such as duplexes, hairpin loops, bulges, internal loops etc. However, in natural RNA, bases are limited to the four predominant structures U, C, A, and G and so the number of compounds that can be used for investigation of parameters of base stacking, base pairing and hydrogen bond, is limited. We synthesized different fluoromodifications of RNA building blocks: 1′-deoxy-1′-(2,4,6-trifluorophenyl)-ß-D-ribofuranose (F), 1′-deoxy-1′-(2,4,5-trifluorophenyl)-ß-D-ribofuranose (M) and 1′-deoxy-1′-(5-trifluoromethyl-1H-benzimidazol-1-yl)-ß-D-ribofuranose (D). Those amidites were incorporated and tested in a defined A, U- rich RNA sequence (12-mer, 5′-CUU UUC XUU CUU-3′ paired with 3′-GAA AAG YAA GAA-5’) (Schweitzer, B.A.; Kool, E.T. Aromatic nonpolar nucleosides as hydrophobic isosters of pyrimidine and purine nucleosides. J. Org. Chem. 1994, 59, 7238 pp.). Only one position was modified, marked as X and Y respectively. UV melting profiles of those oligonucleotides were measured.     

Influence of a fluorobenzene nucleobase analogue on the conformational flexibility of RNA studied by molecular dynamics simulations

Chemically modified bases are frequently used to stabilize nucleic acids, to study the driving forces for nucleic acid structure formation and to tune DNA and RNA hybridization conditions. In particular, fluorobenzene and fluorobenzimidazole base analogues can act as universal bases able to pair with any natural base and to stabilize RNA duplex formation. Although these base analogues are compatible with an A-form RNA geometry, little is known about the influence on the fine structure and conformational dynamics of RNA. In the present study, nano-second molecular dynamics (MD) simulations have been performed to characterize the dynamics of RNA duplexes containing a central 1'-deoxy-1'-(2,4-difluorophenyl)-beta-D-ribofuranose base pair or opposite to an adenine base. For comparison, RNA with a central uridine:adenine pair and a 1'-deoxy-1'-(phenyl)-beta-D-ribofuranose opposite to an adenine was also investigated. The MD simulations indicate a stable overall A-form geometry for the RNAs with base analogues. However, the presence of the base analogues caused a locally enhanced mobility of the central bases inducing mainly base pair shear and opening motions. No stable 'base-paired' geometry was found for the base analogue pair or the base analogue:adenine pairs, which explains in part the universal base character of these analogues. Instead, the conformational fluctuations of the base analogues lead to an enhanced accessibility of the bases in the major and minor grooves of the helix compared with a regular base pair.     

The synthesis of some branched-chain-sugar nucleoside analogues.

1-(2,3-Epoxy-5-O-trityl-beta-D-lyxofuranosyl)uracil was treated with a number of carbon nucleophiles. Ethynyl lithium gave 3'-deoxy-3'-ethynyl-5'-O-trityl-ara-uridine, which was reduced to the corresponding 3'-ethenyl compound. Sodium cyanide gave 3'-cyano-3'-deoxy-5'-O-trityl-ara-uridine which upon alkaline hydrolysis gave the corresponding 3'-carboxamido compound. 1,3-Dithian-2-yl lithium gave 3'-deoxy-3'-(1,3-dithian-2-yl)-5'-O-trityl-ara-uridine. The trityl group was removed from each of these compounds by mild acidic hydrolysis. Treatment of 2 with 0.1M H2sO4 and mercury (II) acetate afforded 3'-acetyl-3'-deoxy-ara-uridine which upon reduction with NaBH4 gave 3'-deoxy-3'-(1-hydroxyethan-1-yl)-ara-uridine. Acetylation of 6 yielded 5'-O-acetyl-3'-acetyl-2',3'-didehydro-2',3'-dideoxyuridine which upon reduction with NaBH4 produced a mixture of 5'-O-acetyl-2',3'-didehydro-2',3'-dideoxy-3'-(1-hydroxyethan -1-yl)uridine and 1-(R)[5-(S)-acetoxymethyl-4-(1-hydroxyethan-1-yl)-tetrahydrofuran- 2-yl]- uracil. Reduction of 14 with Raney nickel followed by removal of the trityl group gave 3'-deoxy-3'-methyl-ara-uridine.     

β-d-2'-α-F-2'-β-C-Methyl-6-O-substituted 3',5'-cyclic phosphate nucleotide prodrugs as inhibitors of hepatitis C virus replication: A structure-activity relationship study

The 3',5'-cyclic phosphate prodrug 9-[β-d-2'-deoxy-2'-α-fluoro-2'-β-C-methylribofuranosyl]-2-amino-6-ethoxypurine, PSI-352938 1, has demonstrated promising anti-HCV efficacy in vitro and in human clinical trials. A structure-activity relationship study of the nucleoside 3',5'-cyclic phosphate series of β-d-2'-deoxy-2'-α-fluoro-2'-β-C-methylribofuranosyl nucleoside prodrugs was undertaken and the anti-HCV activity and in vitro safety profile were assessed. Cycloalkyl 3',5'-cyclic phosphate prodrugs were shown to be significantly more potent as inhibitors of HCV replication than branched and straight chain alkyl 3',5'-cyclic phosphate prodrugs. No cytotoxicity and mitochondrial toxicity for prodrugs 12, 13 and 19 were observed at concentrations up to 100μm in vitro. Cycloalkyl esters of 3',5'-cyclic phosphate nucleotide prodrugs demonstrated the ability to produce high levels of active triphosphate in clone-A cells and primary human hepatocytes. Compounds 12, 13 and 19 also demonstrated the ability to effectively deliver in vivo high levels of active nucleoside phosphates to rat liver.     

RNAi activity of siRNAs modified with 2'-aminoalkyl-substituted fluorinated nucleobases

We recently reported that a 1'-deoxy-1'-(4,6-difluoro-1H-benzimidazol-1-yl)-2'-(beta-aminoethyl)-beta-d-ribofuranose nucleoside appears to be a universal nucleoside which does not differentiate between the four natural nucleosides A, C, G, and U in duplexes. Moreover, ribozymes modified with this nucleoside analog showed a better or at least equal catalytic activity relative to Watson-Crick mismatches.[1] Due to these data, we investigated the ability of this compound to tolerate Watson-Crick mismatches in order to avoid HIV escape mutations in RNA interference. The influence of this nucleoside analog on siRNA efficiency was analyzed with a proven siRNA targeting GFP.     

Synthesis of 3'-deoxy-3'-[4-(pyrimidin-1-yl)methyl-1,2,3-triazol-1-yl]thymidine via 1,3-dipolar cycloaddition

The synthesis of new 3'-deoxy-3'-[4-(pyrimidin-1-yl)methyl-1,2,3-triazol-1-yl]-thymidine 6a-f, from 3'-azido-3'-deoxy-5'-O-monomethoxytrityl-thymidine is described. The key step is the 1,3-dipolar cycloaddition between the azido group of the protected AZT 3 and N-1-propargylpyrimidine derivatives 2a-f. All new derivatives 6a-f were evaluated for their inhibitory effects against the replication of HIV-1 (IIIB), HIV-2 (ROD). No marked activity was found.     

Synthesis of 3'-thioamido-modified 3'-deoxythymidine-5'-triphosphates and their use as chain terminators in Sanger-DNA sequencing

The thioamide derivatives 3'-deoxy-5'-O-(4,4'-dimethoxytrityl)-3'-[(2-methyl-1-thioxo- propyl)amino]thymidine 1 and 3'-deoxy-5'-O-(4,4'-dimethoxytrityl)-3'-((6-([(9H-(fluo-ren-9- ylmethoxy)carbonyl]-amino)-1-thioxohexyl)amino) thymidine 2 were synthesized by regioselective thionation of their corresponding amides 7 and 8 with 2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane-2,4-disulfide (Lawesson's reagent). The thioamides were converted into the corresponding 5'-triphosphates 3 and 4. Compound 3 was chosen for DNA sequencing experiments and 4 was further labelled with fluorescein.     

Inhibition of chloramphenicol binding to Escherichia coli 70S ribosomes by 2'(3')-O-aminoacyl-dinucleoside phosphates derived from the aminoacyl-tRNA acceptor terminus.

The effect of 2' and 3'-O-aminoacyl-dinucleoside phosphates cytidylyl(3'-5')-2'(3')-O-L-phenyl-alanyladenosine (I), cytidylyl(3'-5')-3'-deoxy-2'-O-L-phenylalanyladenosine (IIa), cytidylyl(3'-5')-2'-deoxy-3'-O-L-phenylalanyladenosine (IIIa), cytidylyl(3'-5')-3'-deoxy-2'-O-glycyladenosine (IIb), cytidylyl(3'-5')-2'-deoxy-3'-O-glycyladenosine (IIIb), cytidylyl(3'-5')-3'-deoxy-2'-O-L-leucyladenosine (IIc), cytidylyl(3'-5')-2'-deoxy-3'-O-L-leucyladenosine (IIIc), cytidylyl(3'-5')-3'-O-L-phenylalanyladenosine (IIId) as analogs of the 2'(3')-aminoacyl-tRNA termini, on chloramphenicol binding to 70S Excherichia coli ribosomes was investigated. The association constants (Kb) of the investigated compounds were determined by the equilibrium dialysis method. Based on the constancy of Kb over the range of inhibitor concentration, it was determined that the binding site of the 2' isomers IIa-IIc overlaps with the chloramphenicol site, whereas the variability of Kb for the 3' isomers IIIb, IIIc and especially IIIa seems to indicate that they do not achieve a complete fit. The consistently higher values of the Kb values for the 3' isomers IIIa-IIIc relative to that of the 2' isomers IIa-IIc also indicate a stabilization of the binding of the former due to a specific interaction between its amino acid portion and a ribosomal site.     

Synthesis of 3'-substituted-2',3'-deoxy-2'-methylidenepyrimidine nucleosides.

2'-deoxy-2'-methylideneuridine derivative 9 was converted into 2',3'-didehydro-2',3'-dideoxy-2'-phenyl-selenomethyl derivative 16, which was treated with NCS and tert-butyl carbamate to afford 3'-amino derivative 18 via a [2,3]-sigmatropic rearrangement. Treatment of 9 with DAST gave a mixture of 2',3'-didehydro-2', 3'-dideoxy-2'-fluoromethyl derivative 19 and 3'-"up"-fluoro-2'-methylidene derivative 20 in a ratio of 1.5 : 1. On the other hand, when 12 was treated with DAST, 19 and 3'-"down"-fluoro-2'-methylidene derivative 21 were obtained in a ratio of 1 : 1.6. These nucleosides were converted into the corresponding cytidine derivatives 4, 6, and 8, respectively. The reaction mechanisms as well as biological activity of these compounds will also be discussed.     

Inhibitory effect on NO production of phenolic compounds from Myristica fragrans

Three new phenolics: ((7S)-8'-(benzo[3',4']dioxol-1'-yl)-7-hydroxypropyl)benzene-2,4-diol (1), ((7S)-8'-(4'-hydroxy-3'-methoxyphenyl)-7-hydroxypropyl)benzene-2,4-diol (2) and ((8R,8'S)-7-(4-hydroxy-3-methoxyphenyl)-8'-methylbutan-8-yl)-3'-methoxybenzene-4',5'-diol (3), along with four known compounds (4-7) were isolated from the seeds of Myristica fragrans. Their chemical structures were established mainly by 1D and 2D NMR techniques and mass spectrometry. Their anti-inflammatory activity was evaluated against LPS-induced NO production in macrophage RAW264.7 cells.     

Crystal structure of the 2'-5' RNA ligase from Thermus thermophilus HB8

The 2'-5' RNA ligase family members are bacterial and archaeal RNA ligases that ligate 5' and 3' half-tRNA molecules with 2',3'-cyclic phosphate and 5'-hydroxyl termini, respectively, to the product containing the 2'-5' phosphodiester linkage. Here, the crystal structure of the 2'-5' RNA ligase protein from an extreme thermophile, Thermus thermophilus HB8, was solved at 2.5A resolution. The structure of the 2'-5' RNA ligase superimposes well on that of the Arabidopsis thaliana cyclic phosphodiesterase (CPDase), which hydrolyzes ADP-ribose 1",2"-cyclic phosphate (a product of the tRNA splicing reaction) to the monoester ADP-ribose 1"-phosphate. Although the sequence identity between the two proteins is remarkably low (9.3%), the 2'-5' RNA ligase and CPDase structures have two HX(T/S)X motifs in their corresponding positions. The HX(T/S)X motifs play important roles in the CPDase activity, and are conserved in both the CPDases and 2'-5' RNA ligases. Therefore, the catalytic mechanism of the 2'-5' RNA ligase may be similar to that of the CPDase. On the other hand, the electrostatic potential of the cavity of the 2'-5' RNA ligase is positive, but that of the CPDase is negative. Furthermore, in the CPDase, two loops with low B-factors cover the cavity. In contrast, in the 2'-5' RNA ligase, the corresponding loops form an open conformation and are flexible. These characteristics may be due to the differences in the substrates, tRNA and ADP-ribose 1",2"-cyclic phosphate.     

(3R)-3-amino-4-(2,4,5-trifluorophenyl)-N-{4-[6-(2-methoxyethoxy)benzothiazol-2-yl]tetrahydropyran-4-yl}butanamide as a potent dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes

Novel series of 3-amino-N-(4-aryl-1,1-dioxothian-4-yl)butanamides and 3-amino-N-(4-aryltetrahydropyran-4-yl)butanamides were synthesized and evaluated as dipeptidyl peptidase IV (DPP-IV) inhibitors. Derivatives incorporating the 6-substituted benzothiazole group showed highly potent DPP-IV inhibitory activity. Oral administration of (3R)-3-amino-4-(2,4,5-trifluorophenyl)-N-{4-[6-(2-methoxyethoxy)benzothiazol-2-yl]tetrahydropyran-4-yl}butanamide (12u) reduced blood glucose excursion in an oral glucose tolerance test.     

Some novel pyrimidine nucleoside rearrangements effected by diethylaminosulfur trifluoride (DAST).

Pyrimidine nucleosides (or their 5'-aldehydes) when treated with DAST give O2,5'-(fluoro)-anhydronucleosides. If this is prevented by blocking N-3 or O4, the desired 5'-deoxy-5'-(di)-fluoronucleoside is accompanied by the production of a compound resulting from migration of the base following scission of the N-1-->C-1' bond and formation of O2-->C-5'. This is a particular example of a much more general phenomenon, seen when suitably substituted ribofuranoses are treated with DAST.     

New method for the preparation of 3'- and 2'-O-phosphoramidites of 2'- and 3'-difluoromethyluridine derivatives

Hydrogenation of 2'-deoxy-2'-difluoromethylene-5'-O-dimethoxytrityluridine (1) and 3'-deoxy-3'-difluoromethylene-5'-O-dimethoxytrityluridine (7), gave the corresponding 2'- and 3'-difluoromethyluridine derivatives 2a and 8a. Detritylation of compounds 2a, 2b and 8a, 8b resulted in the formation of 1-(2-deoxy-2-C-difluoromethyl-beta-D-arabino-pentofuranosyl)uracil (3a) and 1-(3-deoxy-3-C-difluoromethyl-beta-D-xylo-pento furanosyl)- uracil (9a) as well as corresponding minor isomers 3b and 9b. Compounds 3a and 3b were also obtained from 2'-deoxy-2'-difluoromethylene-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)uridine (4). Finally, phosphitylation of 2a and 8a provided the title 2'- and 3'-O-phosphoramidites 6 and 10.     

An alkaloid, two conjugate sesquiterpenes and a phenylpropanoid from Pachypodanthium confine Engl. and Diels

Two sesquiterpene-trimethoxystyrene conjugates (E)-1-[3'-(4',8'-dimethylnona-3',7'-dienyl)cyclohex-3'-enyl]-2,4,5-trimethoxybenzene (1) and (Z)-1-[3'-(4',8'-dimethylnona-3',7'-dienyl)cyclohex-3'-enyl]-2,4,5-trimethoxybenzene (2), a phenylpropanoid 1,2,4-trimethoxy-5-(1-methoxy-ethyl)-benzene (3), and an aporphine alkaloid N-acetylpachypodanthine (4), were isolated in addition to several known compounds from cyclohexane, dichloromethane and alkaloid extracts from bark of Pachypodanthium confine. The structures of these compounds were established based on the interpretation of their high resolution NMR (HSQC, HMBC, COSY and NOESY) spectral data.     

Spectroscopic properties of various 2''(3'')-O-aminoacyldinucleoside phosphates analogous to the 3'' terminus of AA-tRNA.

Hypochromicity and circular dichroism data are reported for the 2' and 3'-0-aminiacyldinucleoside phosphates cytidylyl-(3'-5')-2'(3')-0-L-phenylalanyl-adenosine, cytidylyl-(3'-5')-2'-deoxy-3'-0-L-phenylalanyladenosine, cytidylyl-(3'-5')-2'-deoxy-3'-0-glycyladenosine, and cytidylyl-(3'-5')-3'-deoxy-2'-0-L-phenylalanyladenosine, all of which can act as analogs of the 3' terminus of AA-tRNA in various partial reactions of protein biosynthesis. Although all these systems have a 2'-OH group in the furanose of the 3'-residue, differences exist in the extent and/or mode of base-base overlap for most of them, except for cytidylyl-(3'-5')-2'(3')-0-L-phenylalanyladenosine and cytidylyl-(3'-5')-3'-deoxy-2'-0-L-phenylalanyladenosine. It is concluded that the biological activity of the above analogs is affected both by the position of the aminoacyl group and the stacking properties of the bases.     

Effect of JH III and substituted oxime ethers on the in vitro protein and RNA synthesis in male accessory reproductive gland (MARG) of Spodoptera litura (F.)

The physiological action of two substituted oxime ethers namely: 4'-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3'-buten-2'(E)-ketoxime-N-O-propylether (compound No. 3) and 4'-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3'-buten-2'(E)-ketoxime-N-O-pentylether (compound No. 34,) were compared with that of JH III in an in vitro assay to monitor the synthesis of RNA and protein in male accessory reproductive gland (MARG) of Spodoptera litura by using 3H-leucine and 3H-uridine, respectively. Both the compounds have stimulated protein synthesis compare to control. Compound No 34 is slightly more effective than JH III in increasing the protein synthesis at physiological concentration of 10(-6) and 10(-5) M. Compound No 3 and JH III have doubled the RNA synthesis and increased the protein synthesis by 1.5 times over the control at 10(-4) to 10(-6) M concentrations. While JH III at 10(-5) M significantly enhanced RNA synthesis, similar effect is produced only at 10(-3) M by compound No 3 and 34.     

Synthesis, biological evaluation and structural determination of beta-aminoacyl-containing cyclic hydrazine derivatives as dipeptidyl peptidase IV (DPP-IV) inhibitors

Inhibitors of dipeptidyl peptidase IV (DPP-IV) have been shown to be effective treatments for type 2 diabetes. A series of beta-aminoacyl-containing cyclic hydrazine derivatives were synthesized and evaluated as DPP-IV inhibitors. One member of this series, (R)-3-amino-1-(2-benzoyl-1,2-diazepan-1-yl)-4-(2,4,5-trifluorophenyl)butan-1-one (10f), showed potent in vitro activity, good selectivity and in vivo efficacy in mouse models. Also, the binding mode of compound 10f was determined by X-ray crystallography.