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

Con’t

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

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?

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

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

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

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

Ribonucleosides Deoxyribonucleosides

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

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

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

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

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

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)

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

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

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

DNA

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!

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)

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

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

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)

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,

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!

More on Ribozymes We have seen examples of self-cleaving ribozymes Riboswitches represent another class of ribozymes: –Regulate gene expression through a structural rearrangement by binding a small metabolite (from pathway) –Small molecule can bind in “pocket” –Usually located near site of gene (protein expression) In absence of metabolite, the initiation signal of protein synthesis is exposed Conformational change through base pairing blocks expression

glmS ribozyme is a riboswitch that is also self-cleaving: GlcN6P , GlcN6P binds ribozyme = cleavage = no synthase made GlcN6P , GlcN6P = ribozyme is inactive = translation occurs = synthase produced Potential drug target???

Nitrogenous Bases Prebiotic world HCN/CN - + NH 3 ? (similarity to chemical synthesis?) Nicotinamide (NAD + /NADH) Structure/Chemistry A, T, U, C, G Pyridine & pyrrole H-bonding The story so far…

Sugars Structure Reactions of Sugars triose, tetrose, pentose, etc. D/L, R/S Projections Redox reactions Reactions with a Nu acetals Oxonium ion formation Anomeric effect Protecting groups/activating groups (i.e., AZT)  vs  Structural determination by NMR (1D & 2D) Prebiotic word formose reaction (polymers of formaldehyde) Modern world thiazolium ion (cofactor)

Sugar + Nitrogenous Base = Nucleosides Modern World Enzymes (later) Prebiotic world Mineral cations (hydrothermal vents?) Nucleotide + phosphate = Nucleotide Prebiotic World Apatite (P) Template-directed synthesis Modern World ATP DNA/RNA polymerase Ribozymes (link?)

Chemical Synthesis of Oligo’s Challenge: Many different functional groups present  we need to use protecting groups –Same concepts of protection & activation that we have already seen in sugar chemistry Automated synthesis: allows molecular biologists to order oligo’s; made by machine Uses solid phase beads, which allows washing with reagents, solvents, etc. –CPG = Controlled Pore Glass

B = Base, protected to make it non-nucleophilic (amine  amide) This protection must be done prior to attachment to bead  3’ OH is ONLY nucleophile to react

Phosphoramidite – relatively stable – mild conditions for synthesis – high selectivity of activation

Repeat Cycle: 1)Deprotect DMT with H + 2)Add B 3 i.Phosphoramidite ii.Couple 5’OH of growing chain 3)I 2 oxidation to P iv 4)Add B 4, etc… Each step goes in 1-2 mins in > 99% yield! Last step is H + deprotection of DMT Then remove of bead (CPG), remove cyanoethyl & benzoyl (on base)  NH 4 OH

Mechanisms? 1) 2) 3) Final product is the oligo, fully deprotected, released from CPG  elutes from column

RNA synthesis: similar, need a 2’-OH protecting group: –Common one: R 3 Si- (“silyl”) What if you need to know the sequence?

Amplification of nucleic acids (PCR): key to molecular biology  DNA + polymerase + dNTPs + 2 templates + rATP  amplify selected target

Once you amplify DNA, how do you know the sequence? Molecules of different size, each terminated by ddN

In order to “read” sequence, need to tag each ddNTP Previously, 32 P was used (radioactive) Now, each ddNTP is tagged with a different chemical dye –  look at color of peak at terminating nucleotide Based on the synthesis of 2,3-deoxyribose (“dideoxy method”):