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4. Chemical Stability of Nucleic Acids

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4. Chemical Stability of Nucleic Acids 4. Chemical stability of nucleic acids NH2 N 8 N H O 2 Heterocycle: stable - O N P O N H except for reactions with alkylating O HO agents, nitrite and radicals Cleavage of the N-glycosidic bond Purinyl-N-glycosides are cleaved by acid HO OH or hydrazine treatment Cleavage of the phosphodiester linkage: RNA: hydrolyzed by 0.3 M KOH DNA: stable Chemical hydrolysis of phosphoesters Comparison: Ester hydrolysis of carboxylic acid O - O HO- O - R O-R´ + O-R´ R O-R´ R OH OH O tetrahedral transition state Acyl cleavage R O- + HO-R´ Proof: Reaction in 18O labeled water O + H2O, H O + R O R OH HO Alkyl cleavage in specific cases only Chemical hydrolysis of phosphoesters 1.Phosphotriester Apical ligand 1 M NaOH OMe O O 24 h, 20 °C Equatorial ligand - P O P MeO OMe MeO P MeO OMe OMe OMe O- + OH MeOH Trimethyl phosphate Dimethyl phosphate Transition state: dsp3-hybrid! Phosphoryl cleavage Chemical hydrolysis of phosphoesters 2.Phosphodiester 1 M NaOH O 100 °C O P P MeO O-Me MeO O- + MeOH O- O- HO- Dimethyl phosphate Methyl phosphate Alkyl cleavage Half life time: 16 days! Chemical hydrolysis of phosphoesters 2.Phosphodiester HO- H Cyclic phosphodiester (ring strain!) H O - O HO O O O P P O- O O-Me O P O - O- O O O- 2-Hydroxyethyl-methyl-phosphate 2-Hydroxyethyl phosphate Δ H = -26.8 kJ/mol Cf: Dimethyl phosphate: -7.5 kJ/ mol Half life time at 25 °C 25 min! Neighboring group participation Anchimeric assistance OH O H+ O OH OH O-Me + P P P O O H2O O O-Me O O O 2-Hydroxyethyl-methyl Ethylene-phosphate phosphate O 70% 30% P O O „Pseudorotation“: Interchange of apical and O-Me equatorial ligands Leaving group may only be displaced from an apical position (longest bond) Alkaline hydrolysis of DNA and RNA DNA: stable (1 h at 100 °C, 1 M NaOH RNA: hydrolyzed at RT in 0.1 M NaOH BASE BASE O BASE BASE O O O O O O O HO- O OH + O O O OH O P HO O P O O O - P O - O O P O O - HO- - - -O O HO 5' + 3'-Phosphat 2'-Phosphat Enzymatic hydrolysis of RNA Ribonucleases Non-specific cleavage or specific cleavage of bases Ribonuclease A: Pyrimidin-Nucleotides (C,U) Ribonuclease T1 (Aspergillus oryzae): Guanine (in syn-conformation) Ribonuclease P (Ribozymes) Ribonuklease A Isolation from bovine pancreas Many crystal structures (124 entries, e.g. pdb-codes: 1AFK, 1H1H, 1QHC, 1RND) First „NMR-structure“, 4 Nobel prizes M: 13.680, 124 amino acids Chemical syntheses: 1969 Merrifield (0.4 mg), 1979 Yajima (3 mg) RNAse A Catalytic centre: His 12 His 119 Lys 41 Mechanism of ribonuclease A reaction RO Base RO Base O O + NH His 12 HN NH O O OH N - O - P O O O P O N + HN NH O Base HO Base HN O O His 119 OH O OH O RO Base O RO Base O + NH HN O O NH N P O OH O - H H O O HO P O N - HN + O HO Base NH O HN HO Base O O OH O OH Mechanism of phosphodiester- cleavage Product: 3`-Phosphate Major steps: Transesterification - hydrolysis General acid-base catalysis (histidine) Ribozymes – catalytically active ribonucleic acids - metalloenzyme Discovered in 1982 2 Groups 35 – 155 Nucleotides: Hammerhead, Hairpin, Hepatitis Delta Formation of 2´,3`-cyclo-phosphates and 5´-OH (analogous to RNAse A) 100- 3000 Nucleotides: RNase P, Group I and II Introns Formation of 5`-phosphate (and 3´-OH) Self modification, except RNase P (processes t-RNA Precursor) Reaction rate enhancement: ~ 1011 Essential: Mg 2+ Ribozyme Group I Intron-Splicing 3‘ 5‘ Intron 3‘ 5‘ Exon 1 P G Exon 2 G 5‘ Intron 3‘ G G Exon 2 Exon 1 OH Intron 5‘ G Exon 2 G Exon 1 OH 3‘ 5‘ Intron 3‘ Exon 1 Exon 2 G G Magnesium: Coordination of O only (Mn 2+ also with N) 6 Ligands (Ca 2+ > 6) pKa of water lowered to 11.4 Smallest catalytically active RNA: UUU Hydrolysis of GAAA in the presence of Mn2+ Weakest codon-anticodon binding interaction Tetrahymena Group I Intron Hammerhead-RNA.
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