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http://www.scq.ubc.ca/ Sequencing by Synthesis • Newer method to sequence whole genomes – Uses allyl protecting group: color PPPO DNA 3'-OH O + B Mixture of dNTPS O allyl polymerase O DNA color O P O O OH B O RXN STOPS! Pd0 (30 s, deprotects allyl moieties) O DNA O P O O OH OH B free to repeat Sequencing by Synthesis Ju, Jingyue et al. (2006) Proc. Natl. Acad. Sci. USA 103, 19635-19640 Copyright ©2006 by the National Academy of Sciences DNA/RNA Analogues • There have been several recent reports of the modification of oligonucleotides • Modifications have included: – Altering nitrogenous base structure – Employing different sugar structures – Modifying the backbone – Or in combination H2N N N O O N N O O OH(H) O P O NH O O N O O O H N OH(H) N O P O O O O N OH(H) N NH2 • Why make oligo analogues? – Structure/activity relationships (i.e., catalytic versatility) – Antisense technology – Insight into evolutionary process? • Synthetic oligos can be designed to bind with itself, DNA, RNA or all of the above • Effects: – H-bonding (base-pairing) – Other types of interactions (i.e. Van de Waals) – Overall shape (i.e., double helix? Hairpin loop?) • How do we examine interactions? – – – – Melting temp. Tm = 40.5 °C (DNA), 42.5 °C (RNA) NMR (as well other spectroscopic methods) Xray crystallography Calculations Antisense Technology • Employs a synthetic oligo that is complementary (antisense) to an mRNA sequence of interest • One of Two main effects can occur: – Translation arrest (no protein) – Recruits RNAase that degrades mRNA • Potential for therapeutic use – Vitravene (acts on CMV virus) Unnatural Base Pairs • Recall the natural base pairing (Watson-Crick) in DNA: D A H H N O H N N N sugar N sugar A N D N N O H A D H G C • If we change the number of or location of donor/acceptor groups, what interactions can occur? For example: Expand the “genetic alphabet” N • Tm measurements vary dramatically due to changes in H-bonding properties & hydrophobicity D D NH2 N N N N A N A N sugar N sugar NH2 Adenine D D H N H D H N H A H N N • DNA polymerase found to work with some modified bases (oligos) H N A N O N O N H D H D N N A N A sugar A A NH2 NH2 D N D sugar sugar • Such structures would be unlikely to form under prebiotic conditions NH2 Cytosine sugar Cytosine A D O N N NH2 N A N N N N sugar NH2 D guanine N sugar A O A Employing Novel Sugars Some examples: O • 5- vs 6- membered ring 5' Base O • Location of phosphodiester bond 1' 4' 2' 3' • Hydroxyl groups (# & stereochemistry) O • Position of base P OH(H) DNA/RNA O 6' 5' 4' O Base O OH 3' 1' 5' 4' O Base 1' 5' 4' 3' 2' O P -D-Xylopyranosyl NA O 6' O 3' 2' P Hexitol NA (HNA) Base O OH 1' 2' O P 6' OH Altropyranosyl-NA 5' O O Base 1' 4' O 3' 2' P Homo-NA O 6' 5' 4' O O Base 3' 2' P Hexitol NA (HNA) 1' • Adopts chair conformation in oligo • Forms helical duplex with RNA (Tm = + 3°C) & with DNA (Tm = + 3°C) • Adding more hydroxyl groups, increases affinity for RNA – Also increases thermal stability Modification of the Backbone • Peptide Nucleic Acid (PNA) – Aminoethyl glycine units linked by a peptide bond – achiral OH OH NH2 NH2 R O HN Base Base N R O O O HO O NH O R NH Base O HN P -O O O O Base R O O N NH R O HO O Base NH -O P O O O OH O Base HO N O CONH2 Protein PNA RNA • Duplex formation – PNA-DNA Tm = 68.8 °C (~20 °C higher than DNA-DNA!) – PNA-RNA Tm = 72.2 °C • PNA has the recognition properties of DNA (can carry genetic information) & has a higher stability than RNA and DNA • Stability? – Lack of phosphate groups → neutral backbone no electrostatic repulsion! – Not recognized by proteases • Did a PNA molecule precede RNA? – Has demonstrated numerous forms: hairpins, triplexes, etc – Simple in structure (i.e., achiral) & stable – Amino building blocks in primordial soup Another Modified Nucleic Acid - GNA OH glycol • Glycol nucleic acid OH • Can nucleic acid be made with such a simple sugar? – Is a ring structure important? – Minimum # of carbons? Synthesis of GNA O DMT-Cl O DMTO HO H N each base (protected) O CN R2N R2N P O P O HO CN Cl DMTO SN2 (inversion @ least hindered centre) N (Base) R & S GNA made DNA synthesizer DMTO N (Base) • Tested duplex formation: – Duplex formation Tm = 63 °C (22 ° higher than same DNA or RNA sequence!) – GNA more stable than DNA! – Demonstrates that cyclic sugar not necessary! • Was ribose in fact the sugar in the first pre-biotic nucleic acids? • Is GNA a pre-RNA candidate? – A glycerol derivative, which is related to triose – Recall, we looked at 3C sugars (i.e., glyceraldehyde) in relation to the prebiotic formation of sugars An example of chemical biology: • Uses biological concepts, DNA structure • Uses chemical ideas → conformation & functional groups • Uses chemical synthesis principles • Makes a non-natural molecule with novel properties • Relates those properties to the natural system • Problem: Still difficult to predict and analyze singlestranded oligonucleotide structures Part II: The Protein World • We’ve seen how early catalysis by e.g. clay, leads to synthesis of more complex structures • These in turn led to catalysis of specific reactions, eventually leading to proteins taking over as the “normal” catalysts RNA world Protein world Questions 1) How were amino acids first formed? 2) Origin of homochirality: relationship between D-sugars & L-amino acids? 3) How did amino acids condense to give peptides in the prebiotic world? 4) Is this related to chemical peptide synthesis 5) Ribosomal peptide synthesis relationships? 6) Can this knowledge be used to evolve better synthetic strategies? How were Amino Acids Formed? • The Urey-Miller Experiment: R H2O + CH4 + NH3 + H2 OH H2N electrical discharge O amino acid The Urey-Miller Experiment • CH4, NH3 & H2 atmosphere + H2O • Water is heated to induce evaporation • Vapor reaches gas & sparks are then fired through atmosphere (simulates lightning) • Atmosphere is then cooled • Water and organic compounds are trapped • Continued experiment for 7 d, then analyzed residue • Results: – Some insoluble material: likely a cyanide-aldehyde polymer – Aqueous residue showed that 10 -15% of carbon had been converted to organic compounds (including amino acids) – Glycine (R=H) was found to be most abundant (least C-C bond forming reactions needed) – 12 of the other proteinogenic amino acids (20 in modern cells) were formed: – These were amino acids (C relative to C=O) OH H2N O • Note: there are >> 400 naturally occurring amino acids, including both enantiomers, -amino acids, etc For example: H2N OH • Gamma-aminobutyric acid • A amino acid O GABA • The amino acids used for protein synthesis are always L and the stereocentre (except gly) is has S configuration • D amino acids do occur & are often found in nonribosomal peptides • Constructed on a nonribosomal peptide synthase (NRPS) • Found primarily in bacteria and fungi • More on these later! Proteinogenic Amino Acids: • Neutral Amino Acids – – – – – Alkyl (Ala, Ile) The amides (Asn, Gln) Alcohol (Ser, Thr) Thiol (Cys) Thioether (Met) • H+ donors (acidic) – Phenol (Tyr) – Acids (Asp, Glu) • H+ acceptors (basic) – Amines (Lys, His, Arg) pH dependence: OH H2N O H3N O O present at isoelectric point -- zwitterion base acid + OH H3N O H2N O O ions -- more soluble AA precipitates at isoelectric point How to show amino acids are formed in the Urey-Miller Experiment? 1) Ninhydrin detection of amino acids separated by electrophoresis: Apply AA mixture paper + apply electric potential soak in buffer The more –ve charge on the AA at a given buffer pH, the faster it moves towards the cathode (+) +ve charged AAs move toward the anode + - origin: neutral AAs remain • After drying, paper is exposed to ninhydrin: O O H2 O OH O OH O Anhydrous -- all 3 carbons have + next to one another O Hydrate is very stable: + + interaction relieved. Normal form of ninhydrin Ninhydrin reacts with amino acids via imine formation: O O O H2N O H N CO2H OH O O O CO2H N OH + H O -CO2 H+ OH N H NH2 OH OH reacts with another molecule of ninhydrin O O N OH O O O O H2O N H+ HO • This condensation product has an extended conjugated system (how many resonance forms can you draw?) • It absorbs light in the visible region (recall that in UV/VIS spectroscopy, max depends on the length of the conjugated system) O O N OH O • if we spray ninhydrin on the paper chromatogram (or on a TLC plate), purple spots develop • Cannot distinguish between amino acids • Alternatively, can separate amino acids by ion exchange chromatography – Separates based on charge – Derivatize with ninhydrin as eluant comes off the column • Subsequent studies of Urey-Miller Exp’t also showed: – Rapid initial formation of HCN & aldehydes – This was followed by a decrease of these products as amino acids were slowly formed – Hypothesized that amino acids were being formed via a previously known method, the Strecker synthesis • Strecker Synthesis: – Developed by Strecker in 1850 to give racemic amino acids – Can still be found in use, with some modifications O + R H NH3 HCN O H2N OH R Strecker Synthesis: mechanism H+ O R + HO NH2 NH IMINE NH3 R H H R H *** recall from prebiotic synthesis of adenine -CN + NH3 R nitrile hydrolysis H COO- NH2 H R CN Racemic amino acid Was this how amino acids formed in the prebiotic world? • Note the pH rate profile: + H O Rate At low pH: plenty of H+ to increase reactivity of aldehyde, but no –CN or NH3 O H H NH4+ NH3 HCN -CN At high pH: all NH3 & -CN, but no H+ to catalyze addition of Nu: to aldehyde pH 4-5 Optimum pH to carry out reaction is around 4-5 → a balance between [catalyst] & [nucleophile] • Note that the Strecker synthesis must give racemic amino acids: achiral starting materials NC- NH Re face R H Si face -CN • Likewise, Urey-Miller experiment gives racemic AAs • How to separate? – Can perform chromatography using chiral solid phase: two enantiomers bind differently to chiral phase because they are diastereomeric: AAR + SPS AAS + SPS AAR SPS AAs SPS Diastereomers separate • The chemistry of imines is also important in natural processes that are still used today: e.g. Another cofactor, PLP, pyridoxal phosphate (Vit B6 derivative) O H O HO O P O N OH PLP • Involved in amino acid metabolism (e.g. His → histamine) Reacts with amino group of amino acids to make imine (see mechanism) • – Fate of imine? a) Lose the acidic proton (), the reprotonate from the opposite face: epimerization N Enz H COOH H2 N O + S H COOH N R H H N PLP R AA :B-Enz N N Carbon is now sp2 enzyme can add H to either side O N N - COOH OH H H R R H Enz N O H2 O N COOH H H COOH H2 N R R R amino acid hydrolysis regenerates PLP & amino acid H + N b) Decarboxylation: AA decarboxylases N N + PLP O AA N O H H N R H N -N O H + H2 N N H N H H R N H H H For example: NH2 PLP OH H2N decarboxylase H2N NH2 O Lysine - H Enz H2 O R R Cadaverine (rotting flesh) R c) Tautomerize & hydrolyze: transamination (transaminase) N N PLP + AA :B-Enz COOH N N - COOH H H H R R Amine of amino acid converted to carbonyl N O N H2O hydrolysis O O OH N H PLP is now in the amine form & can to perform amination reaction H R + NH2 H H e.g. O O O N OH O pyruvic acid + H - N OH R Imine N O OH H R H+ OH NH2 alanine d) Cleave the R group (on -C) of amino acid: e.g. serine (serine hydroxymethyl transferase – SHMT) N PLP H2N + COOH HO COOH N serine H OH N N + H + H2 N COOH O H glycine H N COOH H + H O • the role of PLP is to make an imine, which stabilizes negative charge through delocalization of charge – Allows for cleavage of C-C and C-N bonds N OH P N - COOH O H R O H O HO O P O N PLP OH N O - N OH H R The synthesis of Dilantin also involves imines (expt 7): + H Ph + H OH H2N O + + H O H2N O Ph Ph H N O Ph OH N H Note the two + C's are adjacent + H H N Ph Ph Ph OH H N O N H Dilantin Ph O Ph N H Ph O N O + HO Ph N H N O Ph N