CHEM30231 (2008-9) Lecturer: Dr V. Ramesh

1. CHEM30231 (2008-9)Lecturer: Dr V. Ramesh NMR in Cheminformatics (8 Lectures) 5th Handout, 9.12.08 Contact details: Room: G-018 (MIB building, North Campus) Tel: X-64539 E-mail: vasudevan.ramesh@manchester.ac.uk

2. Structural Properties of DNA

3. DNA Composition • NUCLEOTIDE • Phosphate group • Pentose (deoxyribose) sugar • Heterocyclic base • PROPERTIES • 4 types of bases • Exist in amino and keto forms • Acidic/ Basic atoms • Affinity for hydrogen bonding

4. Secondary Structure • Chain of nucleotides forms a single strand 5’ - 3’ • Hydrogen bonding between bases of 2 single strands brings duplex together • Watson Crick double helix stabilised by base stacking interactions • Pi-Pi stacking – driving force!! • Hydrophobic Interactions • Electrostatic interactions

5. Tertiary Structure

6. Tertiary Structure

7. Physical Methods for Characterising DNA UV Absorption • AT 260/GC 280nm • π*-π* interactions • Melting curve can determine the melting temperature • Linked to stability NMR • Nuclei in different environments • NMR response transfer -Internuclear distances • Sequential assignment • Investigate connectivities Circular Dichroism • Asymmetric molecules interact with plane polarised light • Different curves for secondary structure foldings (αhelices and βsheets) X-ray crystallography • X-ray beam scattered from electrons in crystal • 3D electron density map • Atomic model – positions, bonds, disorder

8. NMR Assignment of DNA Dickerson Dodecamer Misbah Ghafoor School of Chemistry

9. Introduction Double stranded, right handed B-DNA Highly Stable 2-fold symmetry 6 imino protons 12 H1’protons 9

10. Assignment by NOESY Sequential assignment of the protons using 1H-1H Nuclear Overhauser Effect • There are four types of dipolar couplings which can be carried out for assignment in the NMR spectrum of the DNA. • Intranucleotide dipolar couplings • Sequential dipolar couplings between non-exchangeable protons • Interstrand dipolar couplings • Dipolar coupling between exchangeable protons 10

11. 1. Intranucleotide Dipolar Coupling These are the couplings between protons within a nucleotide Intranucleotide couplings between H8 and H2’ H2’ and H3’ H8 and H1’ 5” 2” Guanosine 11

12. 2. Sequential Dipolar Couplings These are the couplings between non-exchangeable protons between nucleotides Sequential couplings between H8- H1’ H8 – H2”/2’ H Adenine and Guanine 12

13. 3. Interstrand Dipolar Couplings These occur between protons within a base pair Interstrand couplings between G1 and C4 i.e. imino to amino G C 13

14. 4. Dipolar Couplings between exchangeable protons These are couplings between exchangeable protons These occur between amino/amino and imino/amino protons Also imino-imino between base pairs G C 14

15. 1H-NMR Spectrum of DNA Dodecamer A G C T5 T6 Amino T 15

16. Assignment Strategy 1H2O NOESY 2ºC Assignment of exchangeable imino/amino protons 2H2O TOCSY and NOESY 25ºC Assignment of non-exchangeable protons for e.g. (H5-H6), (H1’-H2’/H2”-H3’), (H4’-H5’/H5”) 2H2O NOESY 25ºC Sequential Assignment of non-exchangeable protons for e.g.H6/H8-H1’ or H6/H8-H2’ 16

17. NOESY H2O Aromatics-CH3 Aromatics-Sugars Imino-Amino H1’-Sugars Aromatics-Aromatics Imino-Imino 17

18. Assignment of the EXCHANGEABLE protons 18

19. Assignment of the Exchangeable Protons • Imino protons and amino protons are exchangeable protons • Carry out the experiment in 1H2O, low T • Identify NOEs using 1H-1H Overhauser effect AMINO G – H2 C – H4 A - H4 IMINO G – H1 T – H3 19

20. Imino-Imino δ imino protons = 10-15ppm T imino protons appear lower field than G imino protons We can sequentially walk from G1 / 2  G3 G3  G4 G4  T5 T5  T6 20