Radical Reactions with Double Memory of Chirality ( 2 MOC) for the Enantiospecific Synthesis of Adjacent Stereogenic Quaternary Centers in Solution: Cleavage and Bonding Faster than Radical Rotation

The solution photochemistry of bis(phenylpyrrolidinonyl)ketones (R,R)-1b and (S,S)-1b exhibited a remarkably high memory of chirality. Stereospecific decarbonylation to products (R,R)-3b and (S,S)-3b, respectively, occurred with an ee of ca. 80%. The reaction is thought to occur along the single state manifold by sequential Norrish type-I α-cleavage decarbonylation, and radical–radical combination in a time scale that is comparable to that required for the radical intermediate to expose its other enantiotopic face by rotation about an axis perpendicular to that of the p orbital (ca. 3–7 ps). The absolute configuration of a key intermediate and that of ketone (R,R)-1b were determined by single-crystal X-ray diffraction and the ee values of the photochemical products with the help of chiral shift reagent (+)-Eu(tfc)3 and chiral LC-MS/ MS. On the basis of the ee and de values at 25 °C, it could be determined that ca. 70% of the bond forming events occur with double memory of chirality, ca. 21% occur after rotation of one radical to form the meso product (R,S)-3b, and only 9% occur after double rotation to form the opposite enantiomer. This report represents the first example of a doubly enantiospecific Norrish type-I and decarbonylation reaction in solution and illustrates potentially efficient ways to obtain compounds with adjacent stereogenic quaternary centers

Translesion synthesis by AMV, HIV, and MMLVreverse transcriptases using RNA templates containing inosine, guanosine, and their 8-oxo-7,8-dihydropurine derivatives.

Inosine is ubiquitous and essential in many biological processes, including RNA-editing. In addition, oxidative stress on RNA has been a topic of increasing interest due, in part, to its potential role in the development/progression of disease. In this work we probed the ability of three reverse transcriptases (RTs) to catalyze the synthesis of cDNA in the presence of RNA templates containing inosine (I), 8-oxo-7,8-dihydroinosine (8oxo-I), guanosine (G), or 8-oxo-7,8-dihydroguanosine (8-oxoG), and explored the impact that these purine derivatives have as a function of position. To this end, we used 29-mers of RNA (as template) containing the modifications at position-18 and reverse transcribed DNA using 17-mers, 18-mers, or 19-mers (as primers). Generally reactivity of the viral RTs, AMV / HIV / MMLV, towards cDNA synthesis was similar for templates containing G or I as well as for those with 8-oxoG or 8-oxoI. Notable differences are: 1) the use of 18-mers of DNA (to explore cDNA synthesis past the lesion/modification) led to inhibition of DNA elongation in cases where a G:dA wobble pair was present, while the presence of I, 8-oxoI, or 8-oxoG led to full synthesis of the corresponding cDNA, with the latter two displaying a more efficient process; 2) HIV RT is more sensitive to modified base pairs in the vicinity of cDNA synthesis; and 3) the presence of a modification two positions away from transcription initiation has an adverse impact on the overall process. Steady-state kinetics were established using AMV RT to determine substrate specificities towards canonical dNTPs (N = G, C, T, A). Overall we found evidence that RNA templates containing inosine are likely to incorporate dC > dT > > dA, where reactivity in the presence of dA was found to be pH dependent (process abolished at pH 7.3); and that the absence of the C2-exocyclic amine, as displayed with templates containing 8-oxoI, leads to increased selectivity towards incorporation of dA over dC. The data will be useful in assessing the impact that the presence of inosine and/or oxidatively generated lesions have on viral processes and adds to previous reports where I codes exclusively like G. Similar results were obtained upon comparison of AMV and MMLV RTs

Photochemical Control of RNA Structure by Disrupting π‑Stacking

Photolabile nucleotides that disrupt nucleic acid structure are useful mechanistic probes and can be used as tools for regulating biochemical processes. Previous probes can be limited by the need to incorporate multiple modified nucleotides into oligonucleotides and in kinetic studies by the rate-limiting step in the conversion to the native nucleotide. Photolysis of aryl sulfide <b>1</b> produces high yields of 5-methyluridine, and product formation is complete in less than a microsecond. Aryl sulfide <b>1</b> prevents RNA hairpin formation and complete folding of the preQ₁ class I riboswitch. Proper folding is achieved in each instance upon photolysis at 350 nm. Aryl sulfide <b>1</b> is a novel tool for modulating RNA structure, and formation of 5-methyluridine within a radical cage suggests that it will be useful in kinetic studies

Direct Strand Scission in Double Stranded RNA via a C5-Pyrimidine Radical

Nucleobase radicals are the major family of reactive intermediates produced when nucleic acids are exposed to γ-radiolysis. The 5,6-dihydrouridin-5-yl radical (<b>1</b>), the formal product of hydrogen atom addition and a model for hydroxyl radical addition, was independently generated from a ketone precursor via Norrish Type I photocleavage in single and double stranded RNA. Radical <b>1</b> produces direct strand breaks at the 5′-adjacent nucleotide and only minor amounts of strand scission are observed at the initial site of radical generation. Strand scission occurs preferentially in double stranded RNA and in the absence of O₂. The dependence of strand scission efficiency from the 5,6-dihydrouridin-5-yl radical (<b>1</b>) on secondary structure under anaerobic conditions suggests that this reactivity may be useful for extracting additional RNA structural information from hydroxyl radical reactions. Varying the identity of the 5′-adjacent nucleotide has little effect on strand scission. Internucleotidyl strand scission occurs via β-elimination of the 3′-phosphate following C2′-hydrogen atom abstraction by <b>1</b>. The subsequently formed olefin cation radical yields RNA fragments containing 3′-phosphate or 3′-deoxy-2′-ketonucleotide termini from competing deprotonation pathways. The ketonucleotide end group is favored in the presence of low concentrations of thiol, presumably by reducing the cation radical to the enol. Competition studies with thiol show that strand scission from the 5,6-dihydrouridin-5-yl radical (<b>1</b>) is significantly faster than from the 5,6-dihydrouridin-6-yl radical (<b>2</b>) and is consistent with computational studies using the G3B3 approach that predict the latter to be more stable than <b>1</b> by 2.8 kcal/mol

Reactivity and Specificity of RNase T₁, RNase A, and RNase H toward Oligonucleotides of RNA Containing 8‑Oxo-7,8-dihydroguanosine

Understanding how oxidatively damaged RNA interacts with ribonucleases is important because of its proposed role in the development and progression of disease. Thus, understanding structural aspects of RNA containing lesions generated under oxidative stress, as well as its interactions with other biopolymers, is fundamental. We explored the reactivity of RNase A, RNase T₁, and RNase H toward oligonucleotides of RNA containing 8-oxo-7,8-dihydroguanosine (8oxoG). This is the first example that addresses this relationship and will be useful for understanding (1) how these RNases can be used to characterize the structural impact that this lesion has on RNA and (2) how oxidatively modified RNA may be handled intracellularly. 8-OxoG was incorporated into 10–16-mers of RNA, and its reactivity with each ribonuclease was assessed via electrophoretic analyses, circular dichroism, and the use of other C8-purine-modified analogues (8-bromoguanosine, 8-methoxyguanosine, and 8-oxoadenosine). RNase T₁ does not recognize sites containing 8-oxoG, while RNase A recognizes and cleaves RNA at positions containing this lesion while differentiating if it is involved in H-bonding. The selectivity of RNase A followed the order C > 8-oxoG ≈ U. In addition, isothermal titration calorimetry showed that an 8-oxoG–C3′-methylphosphate derivative can inhibit RNase A activity. Cleavage patterns obtained from RNase H displayed changes in reactivity in a sequence- and concentration-dependent manner and displayed recognition at sites containing the modification in some cases. These data will aid in understanding how this modification affects reactivity with ribonucleases and will enable the characterization of global and local structural changes in oxidatively damaged RNA

Synthesis, Thermal Stability, Biophysical Properties, and Molecular Modeling of Oligonucleotides of RNA Containing 2′‑<i>O</i>‑2-Thiophenylmethyl Groups

Dodecamers of RNA [CUACGGAAUCAU] were functionalized with C2′-<i>O</i>-2-thiophenylmethyl groups to obtain oligonucleotides <b>10</b>–<b>14</b> and <b>17</b>. The modified nucleotides were incorporated into RNA strands via solid-phase synthesis. The biophysical properties of these ONs were used to quantify the effects of this modification on RNA:RNA and RNA:DNA duplexes. A combination of UV–vis and circular dichroism were used to determine thermal stabilities of all strands, which hybridized into A-form geometries. Destabilization of the double stranded RNA was measured as a function of number of consecutive modifications, reflected in decreased thermal denaturation values (Δ<i>T</i>_m, ca. 2.5–11.5 °C). Van’t Hoff plots on a duplex containing one modification (<b>10</b>:<b>15</b>) displayed a ca. ΔΔ<i>G</i>° of +4 kcal/mol with respect to its canonical analogue. Interestingly, hybridization of two modified strands (<b>13</b>:<b>17</b>, containing a total of eight modifications) resulted in increased stability and a distinct secondary structure, reflected in its CD spectrum. Molecular modeling based on DFT calculations shed light on the nature of this stability, with induced changes in the torsional angle δ (C5′-C4′-C3′-O3) and phosphate-phosphate distances that are in agreement with a compacted structure. The described synthetic methodology and structural information will be useful in the design of thermodynamically stable structures containing chemically reactive modifications