Assembling Artificial Nucleic Acid Metalloenzymes Based On C-di-AMP And ATP And Research On Their Chiral Catalytic Functions

Artificial metalloenzymes(ArMs)are resulting from embedding the metallic catalysts into the scaffolds of biological molecules.ArMs are merging the features of both homogeneous catalysis and enzymatic catalysis,which are used to expand the reaction repertoire and explore the catalytic functions of the biomolecules.In recent years,the natural chiral scaffolds of DNA have attracted much attention to construct DNA-based ArMs for enantioselective catalysis.The duplex DNA based ArMs has been extensively investigated and achieved several valuable enantioselective transformations in aqueous media.Also,the other DNA motifs such as G-quadruplex,G-triplex,harpin and single-stranded have been investigated as scaffolds to construct DNA-based ArMs.Compared to DNA,RNA is less stable and few examples of RNA-based ArMs have been reported.Moreover,the described nucleic acids-based ArMs always contain several tens to hundreds of nucleotides,thus the fine structures of nucleic acids-based ArMs are still unclear and the reaction mechanisms are lack of investigation.In this thesis,we are going to employ a cyclic dinucleotide of c-di-AMP and ATP as scaffolds to anchor metallic moieties to construct RNA-based ArMs.The resulted RNA-based ArMs will be investigated for enantioselective catalysis and reaction mechanisms.The main contents are as follows:(1)We found that c-di-AMP and Cu2+could be assembled into an RNA-based ArM(c-di-AMP·Cu2+),which is able to catalyze Diels-Alder reactions of aza-chalcones and cyclopentadiene with high reactivity and good enantioselectivity up to 80%ee.The enantioselective catalytic performances of c-di-AMP·Cu2+has been demonstrated to be largely dependent on different c-di-AMP analogues,the ratios of c-di-AMP and Cu2+,additive metal ions and pH values.Compared to the uncatalyzed reaction and Cu2+ions,c-di-AMP·Cu2+ gave rise to 300-fold and 5-fold rate accelerations,respectively,indicating that c-di-AMP and Cu2+ ions are indeed interacting to be a stable RNA-based ArM.(2)We found that c-di-AMP and an achiral copper(?)complex of Cu(dmbpy)(Cu(?)-4,4'-dimethylbipyridine)are supramolecularly assembling to an RNA-based ArM(c-di-AMP/Cu(dmbpy)).The c-di-AMP/Cu(dmbpy)enables to catalyze an asymmetric sulfoxidation of thioethers and hydrogen peroxide with high activity and modest enantioselectivity.The enantioselective catalytic performances of c-di-AMP/Cu(dmbpy)are related to the following factors:different c-di-AMP analogues,different copper complexes,the ratio of c-di-AMP and Cu(dmbpy),alkaline or alkaline earth metal ions and pH values.Based on the binding affinity assays between c-di-AMP and different Cu(II)complexes,the binding constant between c-di-AMP and Cu(dmbpy)showed a highest affinity(kb=5.55 × 105 M-1)that might be related to the enantioselective induction.(3)We constructed single nucleotide-based ArM(ATP·Cu2+)using ATP and Cu2+ions,which could efficiently catalyze a Diels-Alder reaction with quantitative conversions and good enantioselectivities up to 84%ee.The optimization of the reaction conditions showed that the bases in nucleotide triphosphates,the ratios of ATP/Cu2+,additive ions,pH values and temperatures affect significantly to the catalytic performance of ATP·Cu2+.The ATP·Cu2+provides at least one magnitude of rate acceleration compared to the uncatalyzed reaction.Based on the control experiments of ATP analogues,purine moiety,p-and ?-phosphates are identified to be indispensable residues to ATP·Cu2+.After thorough characterizations of UV-Vis titration,electron paramagnetic resonance(EPR)and NMR titration,together with density function theory(DFT)calculations,a fine coordination structure of ATP·Cu2+ is suggested that the Cu2+ ion binds to one N7 atom,one ?-phosphate oxygen atom,one ?-phosphate oxygen as well as hydrogen bonding between 6-amino and y-phosphate oxygen.