The Dual Role of the 2′-OH Group of A76 tRNATyr in the Prevention of d-tyrosine Mistranslation

Aminoacyl-tRNA-synthetases are crucial enzymes for initiation step of translation. Possessing editing activity, they protect living cells from misincorporation of non-cognate and non-proteinogenic amino acids into proteins. Tyrosyl-tRNA synthetase (TyrRS) does not have such editing properties, but it shares weak stereospecificity in recognition of d-/l-tyrosine (Tyr). Nevertheless, an additional enzyme, d-aminoacyl-tRNA-deacylase (DTD), exists to overcome these deficiencies. The precise catalytic role of hydroxyl groups of the tRNA^Tyr A76 in the catalysis by TyrRS and DTD remained unknown. To address this issue, [³²P]-labeled tRNA^Tyr substrates have been tested in aminoacylation and deacylation assays. TyrRS demonstrates similar activity in charging the 2′ and 3′-OH groups of A76 with l-Tyr. This synthetase can effectively use both OH groups as primary sites for aminoacylation with l-Tyr, but demonstrates severe preference toward 2′-OH, in charging with d-Tyr. In both cases, the catalysis is not substrate-assisted: neither the 2′-OH nor the 3′-OH group assists catalysis. In contrast, DTD catalyzes deacylation of d-Tyr-tRNA^Tyr specifically from the 3′-OH group, while the 2′-OH assists in this hydrolysis.     

2′-OH Protection by the p-Nitrophenylethylsulfonyl (NPES) Group in Oligoribonucleotide Synthesis

Abstract

The NPES group has been sucessfully applied for protection of the 2′-hydroxyl function in synthesizing the adenylate dimer ApA.     

Relative Reactivity of Ribosyl 2′-OH vs. 3′-OH in Concentrated Aqueous Solutions of Phosphoimidazolide Activated Nucleotides

Phosphoimidazolide activated ribomononucleotides (*pN, see structure) are useful substrates for the non-enzymatic synthesis of oligonucleotides. In the presence of metal ions, aqueous solutions of *pN yield primarily the two internucleotide-linked (pN^2'pN and pN^3'pN) and the pyrophosphate-linked (N^5'ppN) dimers. Small amounts of cyclic dimers and higher oligomers are also produced. In this study the relative reactivity of 2-OH vs. 3-OH was determined from the ratio of the yields of pN^2'pN vs. pN^3'pN. Experiments were performed at 23 °C in the range 7.2 pH 8.4 with substrates that differ in nucleobase (guanosine (G), cytidine (C), uridine (U), and adenosine (A)) and leaving group (imidazole (Im), 2-methylimidazole (2-MeIm) and 2,4-dimethylimidazole (2,4-diMeIm)). Two metal ions (Mg²⁺ or Mn²⁺) were employed as catalysts. The conditions used here, i.e. a substrate concentration in the range 0.1 M to 1.0 M and metal ion concentration in the range 0.05 M to 0.2 M, favor base-stacking interactions. The ratio pN^2'pN: pN^3'pN = 2-5: 3-5 was found independent of nucleobase and typically varied between 2 to 3 indicating that the 2-OH is about 2 to 3 times more reactive than the 3-OH. *pN with Im, compared to 2-MeIm and 2,4-diMeIm leaving group, produce lower yields of internucleotide linked dimers, and a higher pN^2'pN: pN^3'pN ratio. Trends in the data, observed with all three leaving groups, suggest an increase in pN^2'pN: pN^3'pN ratio with decreasing substrate concentration (up to 5.47 with 0.051 M ImpG). The observations are in accord with earlier studies reporting a relative reactivity 2'-5': 3'-5'= 6 to 9 obtained with Im as the leaving group, in dilute nucleotide solutions and under conditions that disfavor stacking. It is speculated that the concentration induced change in the relative reactivity is the result of self-association via base-stacking that enhances selectively the proximity of the 3-OH of one molecule to the reactive P-N bond of an other molecule. The implication of these conclusions for oligomerization/ligation reactions is discussed.     

Synthesis of 2′-Substituted Inosine Analogs via Unusual Masking of the 6-Hydroxyl Group

2′-Modified inosine analogs have been synthesized from 6-chloropurine riboside via 6-dimethylaminopurine or 6-benzyloxypurine intermediates. The dimethylaminopurine intermediate was obtained via an unusually facile dimethylamine transfer from dimethylformamide.

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Determination of the Group Electronegativity of CF3 Group in 3′-O-CF3-Thymidine by 1-NMR

Abstract

The interplay of enthalpy of the gauche effect (ΔH°_GE) of the [X3′-C3′-C4′-O4′] fragment in various 3′-substituted (X) 2′,3′-dideoxythymidine derivatives 1–7 and the inherent anomeric effect drives the two-state North ? South equilibrium in the constituent sugar moiety. The group electronegativity of 3′-OCF₃ substituent in Marriott's, Inamoto's and Mullay's scales has been determined from simple calibration graphs correlating the group electronegativity of various 3′-substituents (X) in 2′,3′-dideoxythymidine derivatives 1–7 with the experimental strength (ΔH°_GE) of the [X3′-C3′-C4′-O4′] gauche effect. ΔH°_GE has been experimentally determined from pseudorotational analyses of temperature-dependent ³J_HH coupling constants, and can be used as an unambiguous tool for direct experimental estimation of the group electronegativity of a specific substituent covalently attached to 3′-carbon of 2′,3′-dideoxythymidine, which can be compared, in turn, with the theoretical estimation carried out according to Marriott's or Inamoto's procedure. Inconsistency found between theoretical values in Marriott's and Inamoto's scales, on the one hand, and between our experimental estimate and the theoretical value in Marriott's scale, on the other, have been solved by refining the electronegativity scale using our experimental data for 1–7.     

On the Application of t-Butyldimethylsilyl Group in Chemical RNA Synthesis. Part I. 31P NMR Study of 2′-O-t-BDMSi Group Migration During Nucleoside 3′-OH Phosphorylation and Phosphitylation Reactions

Abstract

³¹P NMR spectroscopy has been used for evaluation of 2′-O-t-BDMSi group migration during reactions of suitably protected 3′-OH ribonucleosides with P(V) and P(III) reagents used in major methodologies for oligoribonucleotide synthesis.     

3′/5′-Regioselectivity of Introduction of the 9-Fluorenyl-Methoxycarbonyl Group to 2′-o-Tetrahydropyran-2-YL-and 2′-O-(4-Methoxytetrahydropyran-4-YL-)-Nucleosides: Useful Intermediates for Solid-Phase-Rna-Synthesis

Abstract

X-ray structure analysis of the more laevorotatory isomers of 2′-O-tetrahydropyranyl-4N-benzoylcytidine (4b) and of 2′-O-tetrahydropyranyluridine (5b) confirmed their chirality at the satellite anomeric centre C2″ to be S. The other diastereomers (4a resp. 5a) exhibited an unexpected reversal of 3′/5′-regio-selectivity when treated with 9-fluorenylmethoxycarbonyl chloride in pyridine. The X-ray crystallographic results form the basis for a mechanistic proposal.     

Use of a Base-Labile Protected Derivative of 6-Mercaptohexanol for the Preparation of Oligonucleotides Containing a Thiol Group at the 5′-End

Abstract

The preparation of a base-labile (Dnpe) protected derivative of 6-mercaptohexanol is described. The use of the phosphoramidite derivative of this compound improves both yields and the time needed for the preparation of oligonucleotides containing a thiol group at the 5′-end.     

Synthesis of the 2′-,3′-Didehydro-2′-,3′-dideoxy and 2′-,3′-Dideoxy Derivatives of 6-Azauridine and a New Route to 2′-,3′-Didehydro-2′-,3′-dideoxy-5-chlorouridine

Abstract

The 5′-O-(4,4′-dimethoxytrityl) and 5′-O-(tert-butyldimethylsilyl) derivatives of 2′-,3′-O-thiocarbonyl-6-azauridine and 2′,3′-O-thiocarbonyl-5-chlorouridine were synthesized from the parent nucleosides by reaction with 4, 4′-dimethoxytrityl chloride and tert-butyldimethylsilyl chloride, respectively, followed by treatment with 1,1′-thiocarbonyldiimidazole. Introduction of a 2′-,3′-double bond into the sugar ring by reaction of the 5′-protected 2′-,3′-O-thionocarbonates with 1, 3-dimethyl-2-phenyl-1, 3, 2-diazaphospholidiine was unsuccessful, but could be accomplished satisfactorily with trimethyl phosphite. Reactions were generally more successful with the 5′-silylated than with the 5′-tritylated nucleosides. Formation of 2′-,3′-O-thiocarbonyl derivatives proceeded in higher yield with 5′-protected 6-azauridines than with the corresponding 5-chlorouridines because of the propensity of the latter to form 2,2′-anhydro derivatives. In the reaction of 5′-O-(tert-butyldimethylsilyl)-2′-,3′-O-thiocarbonyl-6-azauridine with trimethyl phosphite, introduction of the double bond was accompanied by N³-methylation. However this side reaction was not a problem with 5′-O-(tert-butyldimethylsilyl)-2′-, 3′-O-thioarbonyl-5-chlorouridine. Treatment of 5′-O-(tert-butyldimethylsilyl)-2′-, 3′-didehydro-2′-,3′-dideoxy-6-azauridine with tetrabutylammonium fluoride followed by hydrogenation afforded 2′-,3′-dideoxy-6-azauridine. Deprotection of 5′-O-(tert-butyldimethylsilyl)-2′-, 3′-didehydro-2′-,3′-dideoxy-5-chlorouridine yielded 2′-,3′-didehydro-2′-,3′-dide-oxy-5-chlorouridine.     

Synthesis and Antitumor Activity of Acyl and Benzyl Types of Prodrugs of 2′-Deoxy-2′-methylidenecytidine

Abstract

For the purpose of improvement of the in vivo antitumor activity of 2′-deoxy-2′-methylidenecytidine (DMDC, 1), we synthesized its various acyl and benzyl derivatives and evaluated them for their antitumor activity against P388 murine leukemia in mice. In terms of minimum effective dose (30% increase in life span), 5′-O-stearoyl DMDC showed two-fold higher antitumor activity than DMDC on a molar basis, when intraperitoneally (i.p.) administered to mice once a day. The antitumor activities of some other acyl derivatives were almost comparable to that of DMDC, while benzyl derivatives had no antitumor activity. Results on the hydrolysis of 5′-O-acyl derivatives by porcine liver esterase showed that at least these derivatives should not be resistant to enzymatic hydrolysis for exhibiting antitumor activity. After either an i.p. or oral dose of 3′-O-benzyl DMDC, very low concentrations of blood DMDC were seen compared with those after administration of DMDC, suggesting that the inactivity of benzyl derivatives as prodrugs was due to the minimal level of DMDC in circulation after administration.     

Steric Effect of a Bulky Substituent At 5′-Position on the Regioselectivity of 2′ vs. 3′-O-Methylation of N6- Cyclohexyladenosine

Abstract

Steric effect of a trityl or substituted trityl group at 5′-OH of N⁶-cyclohexyladenosine on the regioselective 2′ vs. 3′-O-methylation under phase transfer catalysis conditions was investigated. Compound 4 showed only a modest increase in the selectivity of 2′ over 3′-O-methylation compared to compound 1.     

Synthesis of Oligoribonucleotides Containing 2′-O-Methoxymethyl Group by the Phosphotriester Method

An effective procedure for the synthesis of ribonucleotide monomers containing a 2 ′-О-methoxymethyl-modifying group was developed. These monomers were used for the synthesis of RNA fragments by the solid-phase phosphotriester method under O-nucleophilic intramolecular catalysis. The properties of 2 ′-О-methoxymethyl-containing oligoribonucleotides were examined.     

Unusual Role of the 3-OH Group of Oligosaccharide Substrates in the Mechanism of Bacillus 1,3-1,4-β-glucanase

Abstract

In the mechanism of retaining β-glycosidases, the 2-hydroxyl group of the substrate in the monosaccharyl unit involved in catalysis (subsite -1) is beleived to play an important role through hydrogen bonding interactions with protein residues that are optimized at the transition state. Commonly, removal of the 2-OH group of the substrate results in a 10–12 kcal·mol^-1 transition state destabilization. However, this effect seems not to be general as reported here for Bacillus 1,3-1,4-β-glucanase, a family 16 retaining endo-glycosidase. A p-nitrophenol 2-deosxy tetrasaccharide substrate was synthesized to probe the involvement of the 2-OH group in catalysis. Comparative kinetics with wild-type and subsite +1 mutants show that the 2-deoxy analog is a better substrate than the corresponding 2-hydroxy substrate. It is tentatively proposed that the 2-deoxy analog adopts a different conformation upon binding that compensates for the lack of the 2-OH substituent.     

The Crystal and Molecular Structure of the Complex of 2′3′-Didehydro-2′3′-Dideoxyguanosine with Pyridine

Abstract

The structure of 2′,3′-didehydro-2′,3′-dideoxyguanosine was determined by X-ray crystallographic analysis of the complex with pyridine. The two independent nucleoside molecules have similar, commonly observed glycosyl link (x = -102.3° and -94.2°) and 5′-hydroxyl (y = 54.0° and 47.6°) conformations. The five-membered rings are very planar with r.m.s. deviations from planarity of less than 0.015 A. 2′,3′-Didehydro-2′,3′-dideoxyadenosine has a similar glycosyl link conformation but a different 5′-hydroxyl group orientation and a slightly less planar 5-membered ring.     

Stannylation Approach to the Synthesis of 2′- and 3′-Substituted Analogues of 2′,3′-Didehydro-2′,3′-dideoxynucleosides

Abstract

Three methods are described for the introduction of a tributylstannyl group to the sp²-carbon of 2′,3′-didehydro-2′,3′-dideoxy nucleosides (d44Ns). The resulting stannylated products serve as versatile intermediates for the synthesis of d4Ns having various types of carbon-substituent.     

Introduction of 2-O-benzyl abasic nucleosides to the 3′-overhang regions of siRNAs greatly improves nuclease resistance

Chemically modified siRNAs containing 2-O-benzyl-1-deoxy-d-ribofuranose (R^H_OBn) in their 3′-overhang region were significantly more resistant towards serum nucleases than siRNAs possessing the natural nucleoside in this region. The knockdown efficacies and binding affinities of these modified siRNAs to the recombinant human Argonaute protein 2 (hAgo2) PAZ domain were comparable with that of siRNA with a thymidine dimer at the 3′-end.     

Modulating the regioselectivity of a Pasteurella multocida sialyltransferase for biocatalytic production of 3′- and 6′-sialyllactose

Several bacterial sialyltransferases have been reported to be multifunctional also catalysing sialidase and trans-sialidase reactions. In this study, we examined the trans-sialylation efficacy and regioselectivity of mutants of the multifunctional Pasteurella multocida sialyltransferase (PmST) for catalysing the synthesis of 3′- and 6′-sialyllactose using casein glycomacropeptide as sialyl-donor and lactose as acceptor. The mutation P34H led to a 980-fold increase in α-2,6-sialyltransferase activity (with cytidine-5′-monophospho-N-acetylneuraminic acid as donor), while its α-2,3-sialyltransferase activity was abolished. Histidine in this position is conserved in α-2,6-sialyltransferases and has been suggested, and recently confirmed, to be the determinant for strict regiospecificity in the sialyltransferase reaction. Our data verified this theorem. In trans-sialidase reactions, the P34H mutant displayed a distinct preference for 6′-sialyllactose synthesis but low levels of 3′-sialyllactose were also produced. The sialyllactose yield was however lower than when using PmST_WT under optimal conditions for 6′-sialyllactose formation. The discrepancy in regiospecificity between the two reactions could indicate subtle differences in the substrate binding site in the two reactions. In contrast, the two mutations E271F and R313Y led to preferential synthesis of 3′-sialyllactose over 6′-sialyllactose and the double mutant (PmST_E271F/R313Y) exhibited the highest α-2,3-regioselectivity via reduced sialidase and α-2,6-trans-sialidase activity. The double mutant PmST_E271F/R313Y thus showed the highest α-2,3-regioselectivity and constitutes an interesting enzyme for regioselective synthesis of α-2,3-sialylated glycans. This study has expanded the understanding of the structure-function relationship of multifunctional, bacterial sialyltransferases and provided new enzymes for regioselective glycan sialylation.     

Synthesis of 6-Substituted Purine 2′-Azido- and 3′-Azido-Deoxypentopyranoses

Abstract

Various 6-substituted purine 3′-(2′-) azido-3′, 4′-(2′, 4′-) dideoxy-β-DL-erythro-pentopyranoses (1) (2) have been prepared and compared in terms of a substituent electronegativity parameter. The nucleoside 1a (R=NH₂) is a good competitive inhibitor of adenosine deaminase.     

Evaluation of the Use of the t-Butyldimethylsilyl Group for 2′-Protection in RNA-Synthesis Via the H-Phosphonate Approach

Abstract

Two model compounds. 1 and 2. have been studied to test the stability of the tbutyldimethylsilyl group towards anhydrous acid, aqueous ammonia and tetrabutylammonlum fluoride In THF. Results of relevance to cleavage and migration of phosphodiesters during deprotection of synthetic RNA will be presented.     

Synthesis of 3′-Fluoro-3′-deoxy-N6-cyclopentyladenosine

Abstract

Starting from 9-(β-D-xylofuranosyl)-6-chloropurine, the title compound was prepared in four steps. Reaction with cyclopentylamine followed by treatment of the 2′-O,5′-O-ditritylated material with diethylaminosulfur trifluoride (DAST), yielded after deprotection the desired compound.