H O Origins of Sugars in the Prebiotic World • One theory: the formose reaction (discovered by Butterow in 1861) H O H Mineral catalysis eg Ca(OH) 2 mixture of sugars, including a small amount of ribose formaldehyde Mechanism?

H O H H O O OH HO H O O O OH paraformaldehyde * H H O n * H H 2 O O slow, very unfavorable OH O O O OH H H O O O OH

Con’t via O O OH H O OH H O O O ene-diolate H OH OH glyceraldehyde OH dihydroxyacetone -OH depolymerise O OH H

glycolaldehyde

: simplest sugar & a catalyst for further rxns H O -O H ene-diloate (enol) Ca(OH) 2 O H H pentoses, hexoses -O OH OH O H H O OH OH OH via ene-diolate H O OH H O OH erythrose/threose

H O OH H O OH erythrose/threose -OH retro-aldol 2x O OH H glycolaldehyde: cycle back for catalysis

• Today, similar reactions are catalyzed by thiazolium, e.g., Vitamin B 1 (TPP), another cofactor: • Cf Exp. 7: Benzoin (PP) condensation H O S H • e.g.

N + Py OH H O glycolaldehyde + H O H O OP G3

P

Mechanism? Uses thiazolium OH OH O H O OP D-xylulose-5-

P

H O S N + Py H OH R S N + Py carbanion: zwitterionic; stablized by +/- charge interaction H O OH R S N + Py N + acidifies H OH H OH

:B

thiazolium anion catalyst regenerated R S N + Py R S N + Py H O OH OH OH OP + H O O OH H O OP xylulose-5-P OP H O O H R S N Py OH OH enamine

• 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: CO 2 H RO CO 2 H O + N CO 2 H quinolinate NADH N CO 2 H OH OH RO O OH OH OPP R = H or

P

RO O + OH OH NH 3 RO O + NH 2 nucleosides OH OH Could this process have occurred in the prebiotic world?

• H O Reaction of an oxonium ion with a nitrogenous base: NUCLEOSIDES!

OH O OH OH &/or apatite (mineral phosphate) M n+ Mineral days?

Hydrothermal vents?

H O + O O N H NH O H O O O N NH O H O O O O P OH O OH OH OH OH Thymidine (a nuclesoside) • OH OH Activated leaving group: CATALYSIS 1) 2) Nucleosides are quite stable: Weaker anomeric effect: N< O < Cl (low electronegativity of N) N lone pair in aromatic ring  hard to protonate

1) H O O O N NH O OH OH H O O OH + O OH N NH O Charge separation:unfavorable, since -ve charge is on N, a less electronegative group 2) Anomeric effect: Cl > O > N (remember the glycosyl chloride prefers Cl axial H O O O N NH O H + X H O O O NH + N H O OH OH lone pair part of aromatic sextet OH OH aromaticity destroyed (i.e., pyridine & pyrrole)

• 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: Cytosine Uracil Adenine Guanine + + + + Ribose Ribose Ribose Ribose     Deoxyribonucleosides • Contain 2-deoxyribose, found in DNA Cytidine (C) Uridine (U) Adenosine (A) Guanosine (G) Cytosine Thymine Adenine Guanine + + + + 2-dR 2-dR 2-dR 2-dR     2 ’-deoxycytidine (dC) 2 ’-deoxythymidine (dT) 2 ’-deoxyadenosine (dA) 2 ’-deoxyguanosine (dG)

Ribonucleosides NH 2 N H O O N O H O O N H O N O OH OH cytidine (C) OH OH uridine (U) H O N O N NH 2 N N H O N O N O N N NH 2 OH OH adenosine (A) OH OH guanosine (G) Deoxyribonucleosides NH 2 O N N H H O O N O H O O N O OH 2'-deoxycytidine (dC) OH 2'-deoxythymidine (dT) H O N O N NH 2 N N H O N O N O N N NH 2 OH 2'-deoxyadenosine (dA) OH 2'-deoxyguanosine (dG)

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 H O O A

P

-O O P OH O O A H O O P O O O P O O OH OH OH OH

Adenosine-5' monophosphate (AMP)

O A OH OH

Adenosine-5' diphosphate (ADP)

H O O P O O O P O O O P O O

Adenosine-5' triphosphate (ATP)

O A OH OH Energy source for cell Central to metabolism In the pre-RNA world, how might this happen?

Observation: O H O 5' O 4' 3' OH N 2' 1' OH NH O clay (apatite) 5' phosphate + 3' phosphate + higher phosphates (30 % + 50%) NUCLEOTIDES!

• Surprisingly easy to attach phosphate without needing an enzyme – One hypothesis: cyclo-triphosphate (explains preference for triphosphate O O O P P O O O O P O O H O O T ATP Primary OH?

sterics?

OH OH release of some ring strain in cylco triphosphate drives reaction?

• 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 H O O – Enzyme regulation: Carbohydrate P O O O P O O O P O O O A O O P O OH + ADP OH OH Mg 2+ OH

• 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)  P  P  P PPPO O O O O O B + H O P O P O P O Nuc O O O OH OH Mg 2+ "oligo" (polymer) trinucleotide PPPO O O B 1 O O P O O OH O B 2 a dinucleotide-5'-PPP OH OH

Note that nature faces some problems: 1) 2) Nucleophilic attack required by 3’-OH, not 2’-OH 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

tRNA

Copyright 2006, John Wiley & Sons Publishers, Inc.

DNA

Template-Directed Synthesis in the Pre Biotic Soup

OH H O O N NH O H 2 N N O O O P O OH O O OH OH N O NH O H 2 N N N N N N N N O OH O O P O O O OH OH • 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

RO B 1 O DNA template strand PPPO OH OH O B 2 OH H PP DNA template strand RO O B 1 O O P O OH O O B 2 OH H

Mechanism of Chain Elongation reaction catalyzed by DNA polymerase

RO O B 1 template strand PPPO OH H O B 2 OH H PP RO O B 1 O O P O H O O B 2 OH H

• 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) Yeast tRNA 3' 5' 5' O O B 17 -O Pb 2+ O O P O O O H

C 60 U 59

O O B 17 H O Pb 2+ O O P O O HO

C 60 U 59

3'

• This reaction is specific: – Pb 2+ binds to U 59 /C 60 (if these are mutated  – Cleavage is specific  requires 2’-OH at B 17 no binding) – 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

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!