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-beta-D-ribofuranose (B), 1'-deoxy-1'-(4-fluorophenyl)-beta-D-ribofuranose (4 FB), 1'-deoxy-1'-(2,4-difluorophenyl)-beta-D-ribofuranose (2,4 DFB), 1'-deoxy- 1'-(2,4,5-trifluorophenyl)-beta-D-ribofuranose (2,4,5 TFB), 1'-deoxy- 1'-(2,4, 6-trifluorophenyl)-beta-D-ribofuranose, 1'-deoxy- 1'-(pentafluorophenyl)-beta-D-ribofuranose (PFB), 1'-deoxy-1'-(benzimidazol-1-yl)-beta-D-ribofuranose (BI), 1'-deoxy-1'-(4-fluoro-1H-benzimidazol-1-yl)-1-beta-ribofuranose (4 FBI), 1'-deoxy- 1'-(6-fluoro- 1H-benzimidazol-1-yl)-beta-D-ribofuranose (6FBI), 1'-deoxy- 1'-(4, 6-difluoro- 1H-benzimidazol- 1-yl)-beta-D-ribofuranose (4,6 DFBI), 1'-deoxy- 1'-(4-trifluoromnethyl- H-benzimidazol-1-yl)-beta-D-ribofuranose (4 TFM), 1'-deoxy-1'-(5-trifluoromnethyl-1H-benzimidazol-1-yl)-beta-D-ribofuranose (5 TFM), and 1'-deoxy-1'-(6-trifluoromethyl-1H-benzimidazol-1-yl)-beta-D-ribofuranose (6 TFM). These amidites were incorporated and tested in a defined A, U-rich RNA sequence (12-mer, 5-CUU UUCXUU 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

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.     

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.     

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 5'-functionalized nucleosides: S-Adenosylhomocysteine analogues with the carbon-5' and sulfur atoms replaced by a vinyl or halovinyl unit

Adenosine and uridine analogues functionalized with alkenyl or fluoroalkenyl chain at C5' were prepared employing cross-metathesis, Negishi couplings, and Wittig reactions. Metathesis of the protected 5'-deoxy-5'-methyleneadenosine or uridine analogues with six-carbon amino acids (homoallylglycines) in the presence of Grubbs catalysts gave nucleoside analogues with the C5'-C6' double bond. Alternatively, the Pd-catalyzed cross-coupling between the protected 5'-deoxy-5'-(iodomethylene) nucleosides and suitable alkylzinc bromides also provided analogues with alkenyl unit. Stereoselective Pd-catalyzed monoalkylation of 5'-(bromofluoromethylene)-5'-deoxyadenosine with alkylzinc bromides afforded adenosylhomocysteine analogues with a 6'-(fluoro)vinyl motif. The vinylic adenine nucleosides produced time-dependent inactivation of the S-adenosyl-l-homocysteine hydrolases.     

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.     

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.     

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.     

Crystal structures of B-DNA with incorporated 2'-deoxy-2'-fluoro-arabino-furanosyl thymines: implications of conformational preorganization for duplex stability.

The fundamental conformational states of right-handed double helical DNA, the A- and B-forms, are associated with distinct puckers of the sugar moieties. The furanose conformation itself is affected by the steric and electronic nature of the ring substituents. For example, a strongly electronegative substituent at the C2' position, such as in the 2'-deoxy-2'-fluoro ribo furanosyl analogue, will drive the conformational equilibrium towards the C3'- endo type (north). Conversely, the 2'-deoxy-2'-fluoro arabino furanosyl modification with opposite stereochemistry at C2' appears to have a preference for a C2'- endo type pucker (south). Incorporation of 2'-fluoroarabinofuranosyl thymines was previously shown to enhance the thermodynamic stability of B-DNA duplexes. We have determined the crystal structures of the B-DNA dodecamer duplexes [d(CGCGAASSCGCG)]2and [d(CGCGAASTCGCG)]2with incorporated 2'-deoxy-2'-fluoroarabinofuranosyl thymines S (south) at 1.55 A resolution. In the crystal structures, all S residues adopt an O4'- endo conformation (east), well compatible with an overall B-form duplex geometry. In addition to the increased rigidity of S nucleosides, a clathrate-like ordered water structure around the 2'-fluorines may account for the observed larger thermodynamic stability of DNA duplexes containing 2'-deoxy-2'-fluoroarabino thymidines.     

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.     

Expected distance between terminal nucleotides of RNA secondary structures

In "The ends of a large RNA molecule are necessarily close", Yoffe et al. (Nucleic Acids Res 39(1):292-299, 2011) used the programs RNAfold [resp. RNAsubopt] from Vienna RNA Package to calculate the distance between 5' and 3' ends of the minimum free energy secondary structure [resp. thermal equilibrium structures] of viral and random RNA sequences. Here, the 5'-3' distance is defined to be the length of the shortest path from 5' node to 3' node in the undirected graph, whose edge set consists of edges {i, i + 1} corresponding to covalent backbone bonds and of edges {i, j} corresponding to canonical base pairs. From repeated simulations and using a heuristic theoretical argument, Yoffe et al. conclude that the 5'-3' distance is less than a fixed constant, independent of RNA sequence length. In this paper, we provide a rigorous, mathematical framework to study the expected distance from 5' to 3' ends of an RNA sequence. We present recurrence relations that precisely define the expected distance from 5' to 3' ends of an RNA sequence, both for the Turner nearest neighbor energy model, as well as for a simple homopolymer model first defined by Stein and Waterman. We implement dynamic programming algorithms to compute (rather than approximate by repeated application of Vienna RNA Package) the expected distance between 5' and 3' ends of a given RNA sequence, with respect to the Turner energy model. Using methods of analytical combinatorics, that depend on complex analysis, we prove that the asymptotic expected 5'-3' distance of length n homopolymers is approximately equal to the constant 5.47211, while the asymptotic distance is 6.771096 if hairpins have a minimum of 3 unpaired bases and the probability that any two positions can form a base pair is 1/4. Finally, we analyze the 5'-3' distance for secondary structures from the STRAND database, and conclude that the 5'-3' distance is correlated with RNA sequence length.     

3'-Deoxy-3'-fluoro analogs of core trimers of 2-5A: effect on lytic activity of human NK-lymphocytes]

The effect of core trimers, (2'-5')-analogues of oligoadenylic acid containing 9-(3-deoxy-3-fluoro-beta-D-xylofuranosyl)adenine (AF) and 3'-deoxy-3'-fluoroadenosine (AF) in various positions of the oligomer chain, on the lytic activity of human natural killer cells (NK cells) was studied in three different ways. The cellular cytotoxicity was determined using a highly sensitive nonradioactive approach employing a chelate europium-diethylenetriamino-pentaacetic acid complex (Eu-DTPA). It was shown that all fluorodeoxyanalogues enhance the lytic activity of intact NK lymphocytes, which follows from the lysis rate constant k2. At the same time, the substitution of either the central adenosine fragment or (to a greater extent) the 5'-terminal residue of (2'-5')A3 with AF causes a decrease in the number of active NK cells, which, unlike the case of the natural core trimer, leads to a loss of the capacity to increase the activity of NK. By contrast, isomeric ribo-analogues. (2'-5')(AF)A2 and (2'-5')A(AF)A, and trimers with the 2'(3')-terminal nucleotide substituted by AF or AF increased the activity of NK cells with an effectiveness close to or higher than the natural trimer (2'-5')A3. Inasmuch as isomeric xylo- and ribo-3'-deoxy-3'-fluoroanalogues of (2'-5')A3 are stereochemically modified oligomers, the data unambiguously suggest that the spatial structure of these trimers affects the increase in the lytic activity of NK cells.     

The NMR structure of an internal loop from 23S ribosomal RNA differs from its structure in crystals of 50s ribosomal subunits

Internal loops play an important role in structure and folding of RNA and in recognition of RNA by other molecules such as proteins and ligands. An understanding of internal loops with propensities to form a particular structure will help predict RNA structure, recognition, and function. The structures of internal loops 5' 1009CUAAG1013 3'/3' 1168GAAGC1164 5' and 5' 998CUAAG1002 3'/3' 1157GAAGC1153 5' from helix 40 of the large subunit rRNA in Deinococcus radiodurans and Escherichia coli, respectively, are phylogenetically conserved, suggesting functional relevance. The energetics and NMR solution structure of the loop were determined in the duplex 5' 1GGCUAAGAC9 3'/3' 18CCGAAGCUG10 5'. The internal loop forms a different structure in solution and in the crystal structures of the ribosomal subunits. In particular, the crystal structures have a bulged out adenine at the equivalent of position A15 and a reverse Hoogsteen UA pair (trans Watson-Crick/Hoogsteen UA) at the equivalent of U4 and A14, whereas the solution structure has a single hydrogen bond UA pair (cis Watson-Crick/sugar edge A15U4) between U4 and A15 and a sheared AA pair (trans Hoogsteen/sugar edge A14A5) between A5 and A14. There is cross-strand stacking between A6 and A14 (A6/A14/A15 stacking pattern) in the NMR structure. All three structures have a sheared GA pair (trans Hoogsteen/sugar edge A6G13) at the equivalent of A6 and G13. The internal loop has contacts with ribosomal protein L20 and other parts of the RNA in the crystal structures. These contacts presumably provide the free energy to rearrange the base pairing in the loop. Evidently, molecular recognition of this internal loop involves induced fit binding, which could confer several advantages. The predicted thermodynamic stability of the loop agrees with the experimental value, even though the thermodynamic model assumes a Watson-Crick UA pair.     

Inhibition of uridine phosphorylase by pyrimidine nucleoside analogs and consideration of substrate binding to the enzyme based on solution conformation as seen by NMR spectroscopy

Some 3'- and/or 5'-substituted pyrimidine nucleosides, as well as anhydropyrimidine nucleosides, which have no flexibility about the N-glycosidic bond were studied as inhibitors of thymidine phosphorylase and uridine phosphorylase. The conformation of some analogs was also investigated in order to obtain information on substrate binding to the enzyme. The above compounds, including the potential anti-(human immunodeficiency virus) agent, 3'-azido-2',3'-dideoxy-5-methyluridine were not substrates for either thymidine phosphorylase or uridine phosphorylase. (The only exception was arabinofuranosyl-5-ethyluracil, which proved to be a poor substrate for uridine phosphorylase). The phosphorolysis of thymidine by thymidine phosphorylase was slightly or not at all altered by these pyrimidine nucloside analogs. The lowest Ki was obtained in the case of 3'-azido-2',3'-dideoxy-5-methyluridine and the highest in the case of 2'-deoxylyxofuranosyl-5-ethyluracil, when studying the analogs with flexible structure as inhibitors of uridine phosphorylase. The Ki for 2,3'- and 2,5'-anhydro-2'-deoxy-5-ethyluridine was 5-6 orders of magnitude higher than that for 2,2'-anhydro-5-ethyluridine. Competitive inhibition was observed in all cases. For these three molecules computer-aided molecular modelling predicts the following glycosidic torsion angles chi (O4,-C1,-N1-C2): 109 degrees for 2,2'-anhydro-5-ethyluridine, and 78 degrees and 71 degrees for 2,3'- and 2,5'-anhydro-2'-deoxy-5-ethyluridine respectively. These values are corroborated by high-resolution 13C- and 1H-NMR studies. 2'-Deoxy-5-ethyluridine is predicted to have a syn conformation with chi = 46 degrees and delta E about 2.5 kJ/mol over the minimum energy (in anti position, chi = -147 degrees). 1H and 13C data including homonuclear Overhauser enhancements complete the information about the solution conformation. Considering the Ki values obtained, it is likely that substrates of uridine phosphorylase will bind to the enzyme in the same conformation as 2,2'-anhydro-5-ethyluridine. The greater than 30 degrees deviation from the N-glycosidic torsion angle of 2,2'-anhydro-5-ethyluridine results in much higher Ki values.     

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.     

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.     

The crystal and molecular structure of 2'-deoxy-2'-fluoroinosine monohydrate.

The structure of the hydrate of 2'-deoxy-2'-fluoroinosine has been determined by single-crystal x-ray diffraction. The nucleoside crystallizes in space group P2(1)2(1)2(1) with unit cell dimensions, a = 33.291, b = 10. 871, c = 6.897A. There are two nucleosides and two water molecules in the asymmetric unit. The structure was solved by direct methods and refined to a residual R = 0.095. The two independent nucleosides in the asymmetric unit show different conformations about the glycosidic bond, while other structural details are similar. The base orientation to the sugar is syn in molecule A, whereas anti in molecule B. The exocyclic C(4')-C(5') bond conformation defined with respect to C(3')-C(4')-C(5')-O(5') is gauche+ in both molecules A and B. The sugar ring pucker defined by the pseudorotation phase angle P is a twisted conformation in both, C(3')-endo-C(4')-exo with P = 29 degrees in molecule A and C(4')-exo-C(3')-endo with P = 41 degrees in molecule B. It is shown by comparison with x-ray results of other 2'-fluoronucleosides and unmodified nucleosides including inosines that, in addition to a strong preference of the C(3')-endo type pucker, twisted conformations involving C(4')-exo puckering may be one of characteristic features of 2'-fluoronucleosides.     

Conformational studies of (2''-5'') polynucleotides: theoretical computations of energy, base morphology, helical structure, and duplex formation.

A detailed theoretical analysis has been carried out to probe the conformational characteristics of (2'-5') polynucleotide chains. Semi-empirical energy calculations are used to estimate the preferred torsional combinations of the monomeric repeating unit. The resulting morphology of adjacent bases and the tendency to form regular single-stranded structures are determined by standard computational procedures. The torsional preferences are in agreement with available nmr measurements on model compounds. The tendencies to adopt base stacked and intercalative geometries are markedly depressed compared to those in (3'-5') chains. Very limited families of regular monomerically repeating single-stranded (2'-5') helices are found. Base stacking, however, can be enhanced (but helix formation is at the same time depressed) in mixed puckered chains. Constrained (2'-5') duplex structures have been constructed from a search of all intervening glycosyl and sugar conformations that form geometrically feasible phosphodiester linkages. Both A- and B-type base stacking are found to generate non-standard backbone torsions and mixed glycosyl/sugar combinations. The 2'- and 5'-residues are locked in totally different arrangements and are thereby prevented from generating long helical structures.     

Influence of internucleotide phosphate linkage on relative base stacking in 3'-5' and 2'-5' RNA: a circular dichroic spectroscopic study of RNA hexamer AACCUU.

The variations in base stacking interactions of two isomeric RNA hexamers, 3'-5'r (AACCUU) and 2'-5'r' (AACCUU), have been studied using temperature dependent CD spectroscopy. Both RNA hexamers, in single strand form, exhibited a right handed helical sense. Van't Hoff analysis of the CD spectral results, derived from a two state model, gave a higher enthalpy of stacking for 3'-5' RNA than for 2'-5'RNA. The results suggest that 3'-5' linkage in RNA facilitates formation of better helical stacks in relation to an isomeric 2'-5' linkage.