1. Origins of Sugars in the Prebiotic World One theory: the formose reaction (discovered by Butterow in 1861) Mechanism?

2. Con’t

4. Today, similar reactions are catalyzed by thiazolium, e.g., Vitamin B 1 (TPP), another cofactor: Cf Exp. 7: Benzoin condensation e.g. Mechanism? Uses thiazolium

6. We have seen how the intermediacy of the resonance- stablized oxonium ion accounts for facile substitution at the anomeric centre of a sugar What about nitrogen nucleophiles? Many examples: Could this process have occurred in the prebiotic world?

7. Reaction of an oxonium ion with a nitrogenous base: NUCLEOSIDES! Nucleosides are quite stable: 1)Weaker anomeric effect: N< O < Cl (low electronegativity of N) 2)N lone pair in aromatic ring  hard to protonate

8. 1) Anomeric effect: Cl > O > N (remember the glycosyl chloride prefers Cl axial 2)

9. These effects stabilize the nucleoside making its formation possible in the pre-biotic soup Thermodynamics are reasonably balanced However, the reaction is reversible –e.g. deamination of DNA occurs ~ 10,000x/day/cell in vivo –Deamination is due to spontaneous hydrolysis & by damage of DNA by environmental factors –Principle of microscopic reversibility: spontaneous reaction occurs via the oxonium ion

10. Ribonucleosides & Deoxyribonucleosides Ribonucleosides Contain ribose & found in RNA: Deoxyribonucleosides Contain 2-deoxyribose, found in DNA

11. Ribonucleosides Deoxyribonucleosides

12. Important things to Note: Numbering system: –The base is numbered first (1,2, etc), then the sugar (1’, 2’, etc) Thymine (5-methyl uracil) replaces uracil in DNA Confusing letter codes: –A represents adenine, the base –A also represents adenosine, the nucleoside –A also represents deoxyadenosine (i.e., in DNA sequencing, where “d” is often omitted) –A can also represent alanine, the amino acid

13. Nucleoside + phoshphate  nucleotide In the modern world, enzymes (kinases) attach phosphate groups In the pre-RNA world, how might this happen?

14. Observation: Surprisingly easy to attach phosphate without needing an enzyme –One hypothesis: cyclo-triphosphate (explains preference for triphosphate

15. If correct, this indicates a central role for triphosphates of nucleosides (NTPs) in early evolution of RNA (i.e., development of the RNA world) NTPs central to modern cellular biology

16. Triphosphates Triphosphates are reactive –Attack by a nucleophile at P , P  or P  gives a good resonance stabilized leaving group (can also assisted by metal cation) Other examples where phosphorylation is essential include: –Glucose metabolism –Enzyme regulation: Carbohydrate metabolism, Lipid metabolism, receptors

17. If the nucleophile is the 3’-OH group of another NTP, then a nucleic acid is generated: polymer of nucleotides –Oligomers (“oligos”)  short length (DNA/RNA polymers of long length)

18. Note that nature faces some problems: 1)Nucleophilic attack required by 3’-OH, not 2’-OH 2)Specific attack on  P required 3)In a mixture of NTPs, get non-specific sequence 4)Reaction rate is slow

19. Nucleic acids contain a regular array of bases, spaced evenly along a backbone of phosphates & sugars Even spacing allows self-recognition, –i.e., RNA short stretches form in which bases complement one another –tRNA folds into a specific conformation (more about tRNA later) –DNA: strand I and its reverse complement form a regular sequence with bases paired through H-bonds

20. Copyright 2006, John Wiley & Sons Publishers, Inc. tRNA

21. DNA

22. Template-Directed Synthesis in the Pre- Biotic Soup Template-directed synthesis in the pre-biotic world allows AMPLIFICATION due to MOLECULAR RECOGNITION & rate acceleration results: an entropic effect!

23. Now, catalyzed by enzymes: –DNA polymerase makes DNA copy of a DNA template (i.e., replication) –RNA polymerase makes RNA copy of a DNA template (transcription)

24. Mechanism of Chain Elongation reaction catalyzed by RNA polymerase Mechanism of Chain Elongation reaction catalyzed by DNA polymerase

25. Viruses contain –Reverse transcriptase (RT): makes a DNA copy of RNA genome Template strand = RNA, Product = DNA –RNA synthetase: makes an RNA copy of RNA Template strand = RNA, Product = RNA

26. RNA as a Catalyst = Ribozymes Tom Cech & Sid Altman- Nobel Prize (1989) Ribozymes that catalyze many reactions are being discovered –i.e., cleavage of RNA (this is the reverse of synthesis)

27. This reaction is specific: –Pb 2+ binds to U 59 /C 60 (if these are mutated  no binding) –Cleavage is specific  requires 2’-OH at B 17 –One of few systems where x-ray structure is available revealing potential mechanism Another example: Can RNA catalyze addition of a base to a sugar? YES! see (on website): Lau, M; Cadieux, K; Unrau, P. J. Am. Chem. Soc., 126, 15686- 15693

28. Chemical synthesis  random sequences of RNA a)Attach sugar, lacking base, to 3’ end b)Few molecules react with base to make nucleotide at 3’ end c)Sort out those with base at 3’ end d) Amplify (PCR), enrich pool & cycle many times Gives pure catalytic RNA!