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An improved version of the H-phosphonate approach to RNA-synthesis is presented and the studies that led to alterations in the protecting group strategy are discussed.

The structural determination of modified nucleosides is important for understanding the chemistry, structure, and functional changes that they introduce to the nucleic acids in which they occur. Thiolation of transfer RNA wobble position uridine produces an energetically stabilized conformation of the nucleoside in solution at ambient temperature that is independent of the nature of the 5-position substituent and is of biological significance to tRNA selection of only those codons ending in adenosine (Sierzputowska-Gracz, H.; Sochacka, E.; Malkiewicz, A.; Kuo, K.; Gehrke, C.; Agris, P. F. J. Am. Chem. Soc. 1987, 109, 7171-7177. Agris, P. F.; Sierzputowska-Gracz, H.; Smith, W.; Malkiewicz, A.; Sochacka, E.; Nawrot, B. J. Am. Chem. Soc., in press). Dinucleoside monophosphates have been synthesized as models for investigating the conformations and structures of wobble position uridine-34 that is thiolated and differently modified at position-5 and that is either 3′-adjacent to the invariant uridine-33 in tRNA or 5′-adjacent to the second anticodon position uridine-35. The structures and conformations of 11 dinucleoside monophosphates were analyzed by 1H, 13C, and 31P magnetic resonance (NMR) spectroscopy. Within the dinucleosides, the individual modified uridine structures and conformations were very similar to those of their respective mononucleosides. The 2-position thiolation, and not the 5-position modification, produced a significantly more stable, C(3′) endo, gauche+, anti conformer. However, within those dinucleosides in which the 2-thiouridine was 5′ to the unmodified uridine, the nucleic acid backbone torsion angles of the unmodified uridine 5′-phosphate were affected, as determined from the scalar coupling constants J1H1H, J1H31P, and J13C31P. In contrast, uridines that were only 5-position modified did not affect the conformation of the 3′-adjacent unmodified uridine phosphate. The structural data obtained and the nucleoside conformations derived from the data support the "modified wobble hypothesis" (Agris, P. F. Biochimie 1992, 73, 1345-1349); i.e., the tRNA wobble position-34 nucleoside is modified in such a way as to constrain not only its own conformation but also the structural conformation of the anticodon, thereby producing a specific codon selection during protein synthesis.

Hydrolytic removal of the 2'-tert-butyldimethylsilyl (TBDMS) group from a 2'-O-TBDMS protected UpU dimer [U(2'-Si)pU] (1) (Si = TBDMS) and related derivatives under various acidic conditions was studied in detail. First, desilylation of 1 by use of acetic acid was examined. Consequently, we made the unprecedented discovery that cleavage of the 2'-silyl ether linkage occurred fastest at a very low concentration of acetic acid within the range of 5-10%, depending on the temperature. Formic acid could cleave the silyl ether much faster than acetic acid, but the relationship between the reaction rate and the concentration of acid was different from that of acetic acid. The use of 20-40% formic acid resulted in very effective elimination of the 2'-TBDMS group. Moreover, diluted HCl solution (pH 2.0) could cleave the Si-O bond faster than acetic acid at 30°C. In contrast, the 2'-silyl gloup of the corresponding methylphosphonate derivative [U(2'-Si)p(Me)U] (3) was much more stable than that of 1. In the case of a diastereomeric mixture of the phosphorothioate dimer [U(2'-Si)psU] (2), a big difference in reaction rate between the Rp- and Sp-diastereomers was observed. These results strongly suggest that neighboring group participation of the 3'-5' phosphorodiester group is involved in the present acid-catalyzed 2'-desilylation. These conditions were successfully applied to the deprotection of the 2'-TBDMS group of an RNA intermediate which was chemically synthesized by the conventional phosphoramidite approach on a CPG gel.

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The catalysis of phosphoryl transfer by metal ions has been intensively studied in both biological and artificial systems, but the status of the transient pentacoordinate phosphoryl species (as transition state or intermediate) is the subject of considerable debate. We report that dinuclear metal ion complexes that incorporate second sphere hydrogen bond donors not only promote the cleavage of RNA fragments just as efficiently as the activated analogue HPNPP but also provide the first examples of metal ion catalyzed phosphate diester isomerization close to neutral pH. This observation implies that the reaction catalyzed by these complexes involves the formation of a phosphorane intermediate that is sufficiently long-lived to pseudorotate. Copyright

A novel method for the synthesis of oligoribonucleotide using 1-(2-cyanoethoxy)ethyl (CEE) as a 2′-hydroxy protecting group has been developed. A novel method for the synthesis of oligoribonucleotides using 1-(2-cyanoethoxy)ethyl (CEE) as a 2′-hydroxy protecting group has been developed. A CEE group was introduced to the 2′-position of N-acyl-3′,5′-O-silyl-protected ribonucleosides under acidic conditions in good yields. The 2′-O-CEE group was found to be stable in an aqueous or ethanolic ammonia and was quickly removed by treatment with anhydrous tetrabutylammonium fluoride (TBAF). A combination of the use of N-acyl and 2′-O-CEE protecting groups enabled a reliable and complete two-step deprotection, first with NH3-EtOH, then with TBAF in THF, without cleavage of internucleotidic linkages.

The comparative rates of acid-catalysed removal often 1-aryl-4-methoxypiperidin-4-yl 8 (R = Me) [including the previously reported Ctmp 5 and Fpmp 6] protecting groups for the 2′-hydroxy functions in oligoribonucleotide synthesis are discussed. These studies have led to the development of the 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep) protecting group 8 (R = Et, R1 = R2 = H, R3 = Cl) which is both more stable than the Ctmp and Fpmp groups at pH 0.5 and more labile at pH 3.75. The influence of the ribonucleoside aglycone on the stability of the 2′-O-Fpmp and 2′-O-Ctmp protecting groups both at low and high pH is examined. The Royal Society of Chemistry 2000.

Metal-ion-promoted hydrolytic reactions of the SP and RP diastereomers of the phosphoromonothioate analogues of uridylyl(3′,5′)uridine [3′,5′-Up(s)U] and their cleavage products, diastereomeric uridine 2′,3′-cyclic phosphates [2′,3′-cUMPS], were followed by HPLC as a function of pH (4.7-5.6) and metal ion concentration (1-10 mmol L-1). With 3′,5′-Up(s)U, three reactions compete: (i) cleavage to 2′,3′-cUMPS, (ii) isomerization to 2′,5′-Up(s)U, and (iii) desulfurization to an equilibrium mixture of 2′,5′- and 3′,5′-UpU. Of these, the cleavage to 2′,3′-cUMPS is markedly accelerated by Zn2+, Cd2+, and Gd3+, the rate enhancements observed with the Sp isomer at [Mz+] = 5 mmol L-1 and pH 5.6 (T = 363.2 K) being 410-, 3600-, and 2000-fold, respectively. The effect of Mn2+ and Mg2+ on the cleavage rate is, in turn, modest (6- and 1.7-fold acceleration, respectively). The rate-accelerations are almost equal with the SP and RP diastereomers. The metal-ion-promoted reaction is first-order in both the hydroxide and metal ion concentration, and it proceeds by inversion of configuration at phosphorus, consistent with an in-line displacement mechanism. The isomerization and desulfurization are much less susceptible to metal ion catalysis: 6.4- and 7.7-fold accelerations were observed with Zn2+, respectively. Gd3+ does not promote these reactions at all. The isomerization proceeds by retention of configuration at phosphorus, consistent with formation of a pentacoordinated thiophosphorane intermediate having the entering 2′-hydroxy group apical and the leaving 3′-hydroxy equatorial, and subsequent pseudorotation posing the leaving group apical. The hydrolysis of 2′,3′-cUMPS is accelerated by metal ions slightly more efficiently than the cleavage of 3′,5′-Up(s)U to 2′,3′-cUMPS. In striking contrast to the reactions of 3′,5′-Up(s)U, the hydrolytic desulfurization to 2′,3′-cUMP is accelerated as efficiently as its endocyclic hydrolysis to uridine 2′and 3′-phosphoromonothioates [2′- and 3′-UMPS]. Somewhat unexpectedly, the latter compounds were observed to undergo metal-ion-promoted cyclization/desulfurization to 2′,3′-cUMP. The hydrolysis of 2′- or 3′-UMPS to uridine was, in turn, observed to be retarded by metal ions. The mechanisms of the partial reactions are discussed.

This paper describes general methods for the synthesis of N- phosphorylated ribonucleosides and oligonucleotides containing a 2'-O- phosphorylated or 2'-O-thiophosphorylated ribonucleoside. The NMR-based conformational analysis and computational molecular dynamics simulation of the 2'-O-phosphorylated ribonucleoside residue in such modified oligonucleotides suggested that the ribose residue existed preferentially in a C2'-endo conformation. It was also found that simple heating of 2'-O- phosphorylated oligonucleotides resulted in rapid dethiophosphorylation.

The hydrolysis of uridylyl(3',5')uridine promoted by Mg2+, Zn2+ and Zn2+aneN3 (aneN3 = 1,5,9-triazacyclodecane) has been studied in imidazole, HEPES and triethanolamine buffers and the origin of marked electrolyte effects observed with Zn2+aneN3 has been examined.The results obtained suggest that the basic buffer constituent does not serve as an external general base, but the catalytic activity of the metal ion species is influenced by the coordination to the buffer base and other Lewis bases present in the solution.The data lend additional support to a bifunctional mechanism, which consist of coordination of the metal ion to the anionic phosphodiester and intracomplex general base (or nucleophilic) catalysis by its hydroxo ligand.

The new type protecting group, 1-(2-chloroethoxy)ethyl (Cee) group has been employed for the protection of the 2'-OH groups of ribonucleoside residues in the synthesis of oligoribonucleotides by the phosphoramidite approach on a solid support, using the acid-labile 5'-O-dimethoxytrityl (DMTr) group.This group is completely stable under the acidic conditions required to remove the 5'-terminal protecting groups in oligonucleotide synthesis on a solid support, and yet is easily removable under mild condition of acidic hydrolysis (pH 2.0) for the final unblocking step.The Cee-protected ribonucleoside 3'-phosphoramidite units were evaluated in the synthesis of a series of oligoribonucleotides consisting of the homopolymers of cytidine, the box 9R and 9R' sequences of Tetrahymena rRNA, and a leader sequence of phage Qβ-A protein mRNA.A full data for the deprotection and purification of synthetic oligoribonucleotides are also described.

A new 2'-hydroxyl protecting group, thio>methyl (MPTM), was introduced via a three-step reaction into the 2'-position of uridine and cytidine units required for the synthesis of oligoribonucleotides in the phosphotriester approach.By using these monomer building blocks, CpUpG was synthesized.

Comparative 1H, 31P NMR and CD spectroscopic results and enzymatic cleavage of single stranded RNA hexamers which have identical base sequence (AACCUU) but are regioisomeric at the internucleotide phosphate linkages (2'-5', 3'-5' and their covalent hybrid) are presented. The 31P NMR results revealed significant differences in local phosphate backbone conformation among these isomers, with 2'-5' isomer exhibitingf maximum heterogeneity as compared to the 3'-5' isomer, the analogous DNA hexamer and the covalent hybrid hexamer. In contrast to this, there are no appreciable differences in the overall base-base stacking as seen in the CD spectra of 2'-5' and 3'-5' isomers. All RNA hexamers were synthesised by solution phase phosphotriester chemistry with t-butyldimethylsilyl (TBDMS) as 2'-O-protecting group. The fully protected oligorobomers were deprotected in two steps: (i) saturated anhydrous MeOH-NH3 for phosphate and amino deprotections and (ii) TBAF for removal of 2'-O-TBDMS group. Use of sat. MeOH-NH3 (instead of aq. NH3) prevents 2'-3' internucleotide phosphate migrations, chain fragmentations and 5'-terminal modifications by neighbouring group participation (NGP). The retention of isomeric integrity and absence of 5'-terminal modification in the final products was established by digestion with several nucleases.

The 1--4-methoxypiperidin-4-yl has an acid lability similar to that of the 4-methoxytetrahydropyran-4-yl (Mthp) protecting group under mild hydrolytic conditions ; however, under the relatively more drastic conditions required for the complete removal of a 9-phenylxanthen-9-yl (Px) group, the Ctmp protecting group remains virtually intact.

A study of four different O-4 and N-3 protected uridine derivatives, 4 to 7, for their stabilities under different conditions versus their abilities to undergo nucleophilic substitution reaction at C-4 by an appropriate oxygen or a nitrogen nucleophile has established a general strategy for the site-specific modification of a particular pyrimidine residue in a model fully protected diuridylic acid to give either UpC, CpC or UpU, depending upon the deprotection condition.

The phosphorylating agent obtained by treatment of S-4-methylphenyl phosphorodichloridothioate with 1-hydroxybenzotriazole can not only be applied for the introduction of polyphosphate functions at the terminal ends of nucleic acids, but also for the formation of 3'-5'-phosphotriester linkages.

Several kinds of organotin compounds involving stannyl oximates have been examined as reagents for deblocking the internucleotidic phenylthio group from an appropriately protected S-phenyl diuridine phosphorothioate (1).Among them, bis(tributyltin) oxide was found to be very effective.

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A series of phosphorodichloridites has been prepared incorporating the most commonly used phosphate protecting groups in oligonucleotide synthesis.The groups incude trichloroethyl, tribromoethyl, cyanoethyl, benzyl, methyl, p-chlorophenyl, and nitrophenetyl.The trichloroethyl and the nitrophenethyl appear to be the most stable groups while the cyanoethyl and methyl offer specific advantages.The benzyl and p-chlorophenyl groups are subject to limitations on their utility.Condensations can be carried out in a range of solvents incuding THF, pyridine, and DMF and at temperatures from -78 deg C to 20 deg C with a slight drop in yield with increasing temperature, at least for dinucleotide condensations.

In the presence of cesium fluoride, the reactions of a fully protected 5'-O-dimethoxytrityl-2'-O-tetrahydropyranyl-N-acylnucleoside 3'-(4-chlorophenyl, 5-chloro-8-quinolyl) phosphates with 5'-hydroxyl nucleosides proceeded rapidly under mild conditions to afford the corresponding ribonucleoside monophosphates in good yields.

Preparative synthesis of dinucleoside monophosphates, catalyzed by ribonuclease A, is described.Uridine 2',3'-cyclic phosphate was used as a donor, the acceptors being uridine (Ia), N3-methyluridine (Ib), 5-methyluridine (Ic), 6-methyluridine (Id), 3-(β-D-ribofuranosyl)uracil (IIa), 1-methyl-3-(β-D-ribofuranosyl)uracil (IIb), 6-azauridine (III) and 6-methyl-2'-deoxyuridine (IV).The obtained compounds of the type UpN (where N is the nucleoside moiety I-IV) were characterized by paper chromatography, electrophoresis and UV-spectra.

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The synthesis of compounds having a phosphite bridge between nucleoside units, their characteristics toward phosphodiesterases, their conversion to normal nucleotides and their use in oligonucleotide synthesis is described.

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