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