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CHEM 460 / 560 Prebiotic Chemistry Dr. Niles Lehman Department of Chemistry Portland State University [email protected] http://web.pdx.edu/~niles/Lehman_Lab_at_PSU/ Chem_460_560.html The Timeline of Life Joyce (2002) Nature 418, 214-221 timelines: ORIGINS OF LIFE ON THE EARTH 15 bya 10 bya 13.7 BIG BANG (see P&G Fig. 2.1) 5 bya 0 bya 4.6 4.0 4.55 origin of the sun 2.5 0.6 eubacteria/ archaea split origins of multicellularity origin of the Earth What is Life? the “list of characteristics” approach: • growth • response to stimulus • metabolism • reproduction • evolution LIFE = “a self-sustaining chemical system capable of darwinian evolution” (Joyce/NASA) life non-life the non-life-to-life transition at 4.0 +/– 0.1 billion years ago a “dead” bag of chemicals ??? an “alive” bag of chemicals Lehman: “the origins of life is a chemical problem in a biological context” autocatalysis A + B C autocatalysis is a situation in which the product of a reaction catalyzes its own synthesis from reactants add Mn++ 2MnO4++ + 5H2C2O4 + 6H30+ 2Mn++ + 10CO2 + 14H2O a necessary, but not sufficient, requirement for “life” the chemistry of life the life on the Earth is based on Carbon C atomic number = 6 electronic configuration: 1s2, 2s2, 2p2 atomic mass = 12.011 isotopic abundance on Earth: 11C = 0% (synthetic) 12C = 98.9% 13C = 1.1% 14C = 1 PPT (0.0000000001%) carbon vs. silicon carbon is more suitable for life (self-reproducing and evolving systems) because: • the C-H, C-N, C-O, and C-C bond energies are similar • C-X single, double, and triple bond energies are similar • breaking of the C-H bond requires high ΔEa • carbon dioxide, the oxidative end product, is a gas the stuff of life • proteins (amino acids) • lipids (alcohols & fatty acids) • carbohydrates (sugars) • nucleic acids (nucleotides) • small molecules (water, metals, ions, etc.) all are polymers formed by condensation reactions ...in the “primordial soup”? the elements of life sum = about 22 elements elemental abundances in the universe for our Sun: see P&G, Fig. 1.2; for rocky planets, see P&G, Fig. 1.3 water is the solvent of life water is highest water is high water is lowest the three “stages” in the evolution of life 1. chemical evolution 2. self-organization 3. biological evolution life can be considered a “negentropy machine” hν ΔS < 0 heat 1. Light energy from the sun is absorbed by the Earth and eventually converted into energy that living things can use (ATP). 2.Living thing use this energy and perhaps convert it to other forms of chemical energy, but this conversion is not perfect...some is lost as low-grade energy (heat). 3.Life then, uses the sun’s energy to maintain its own order. 4.Because the environment is constantly changing, life must acquire information from the environment (through sensing devices) and alter its own information content accordingly. 5.Life, therefore, are little pockets of NEGENTROPY, where the order is temporarily greater than its surroundings. The Seven Challenges to a Prebiotic Chemist 1. The origin/source of the elements 2. The origin/source of small molecule precursors 3. The origin/source of monomers 4. The condensation problem 5. The self-replication problem 6. The chirality problem 7. The compartmentalization problem The Seven Challenges to a Prebiotic Chemist 1. The origin/source of the elements 2. The origin/source of small molecule precursors 3. The origin/source of monomers 4. The condensation problem 5. The self-replication problem 6. The chirality problem 7. The compartmentalization problem The Big Bang 13.7 bya the four fundamental forces in Nature strong nuclear force holds nuclear particles together (p + n) >> weak nuclear force >> responsible for radioactive decay (n p + e–) electromagnetic force >> holds electrons to nuclei (CHEMISTRY) gravitational force holds matter together into larger structures from the Big Bang to the formation of our Solar system t = 0 : the Big Bang -- only electrons, neutrons, protons, and photons e–, n, p, hν from the Big Bang to the formation of our Solar system t = 100 sec : temperature cooled below 1 billion K; the strong nuclear force was no longer overwhelmed, and protons and neutrons could combine to form nuclei “Big Bang nucleosynthesis” p = 1H p+p D 3He D+p 3He + 3He 4He + 2p from the Big Bang to the formation of our Solar system t = 377,000 years: temperature cooled below 3000 K; the recombination era the electromagnetic force was no longer overwhelmed, and electrons could remain with nuclei universe anisotropy was key to life! the background microwave radiation in the universe is slightly anisotropic: it does NOT look exactly the same in all directions universe anisotropy was key to life! 10-parts-per-million differences in energetic distributions led to... unequal mass distributions, which led to... clumping of interstellar gasses, which led to... a trillion or so lumps of protogalaxies, inside of which other anisotropies led to... STAR SYSTEM FORMATION (formation of stars = elements, the solar system, & the Earth) accretion protostar accretion discs protoplanets inner, rocky planets outer, gaseous planets nucleosynthesis in the Sun Sun: T = 16 million K the Bethe & Weizsacker carbon cycle distribution of heavier elements via supernovae events elements above atomic number 26 (Fe) come from exploding stars elsewhere planetary formation inner, rocky planets: Cn, Sin, Fe outer, gaseous planets: H2, He, NH3, and CH4 formation of Earth’s moon massive collision at 4.51 +/– 0.01 bya was another key event in the origins of life the history of large impacts on the Earth and Moon moon-formation impact red: impacts on Moon blue: impacts on the Earth habitable zones solar system habitable zone • • • • • • only one star our Sun is relatively massive broad region where liquid water can form Earth is outside tidal lock zone Earth has a moon Jupiter is “out there” galactic habitable zone • • • not too near the galactic center not too far away from the galactic center the Sun’s orbit is circular http://movies.netflix.com/WiMovie/Where_Did_We_Come_From_Nova_scienceNOW/70170758?trkid=496624 The Seven Challenges to a Prebiotic Chemist 1. The origin/source of the elements 2. The origin/source of small molecule precursors 3. The origin/source of monomers 4. The condensation problem 5. The self-replication problem 6. The chirality problem 7. The compartmentalization problem the central dogma of molecular biology Figure 5-21 The central dogma of molecular biology. life: needs all this plus anything else to keep it “safe” the chemical requirements of Life • proteins (amino acids) • lipids (alcohols & fatty acids) • carbohydrates (sugars) • nucleic acids (nucleotides) • small molecules (water, metals, ions, etc.) all are polymers formed by condensation reactions ...in the “primordial soup”? review: elements of life • nucleic acids (CHOPN) • proteins (CHOSN) • lipids (CHO) • polysaccharides (CHO) • catalysts (Fe, Mg, Ca, Mn, Ni, Zn, Cu, Se, Co, Mo) • counterions (Na, K, F, Cl, Br, I) • neutrals, for clays (Al, Si) in total, about 22–24 elements: H, C, N, O, F, Na, Mg, Al, Si, P, S, Cl, K, Ca, Mn, Fe, Co, Ni, Cu, Zn, Se, Br, Mo, I Darwin’s “Warm Little Pond” “It is often said that all the conditions for the first production of a living organism are now present, which could ever be present. But if (and oh! what a big if) we could conceive in some warm little pond with all sorts of ammonia and phosphoric salts, light, heat, electricity, etc., present, that a protein compound was chemically formed ready to undergo still more complex changes, at the present day, such matter would be instantly devoured or absorbed, which could not have been the case before living creatures were formed.” Darwin, 1871, unpublished letter small molecules in interstellar space, as detected by radiotelescopy hydrogen cyanide formaldehyde > 120 organic molecules have been detected to date, mostly by microwave spectroscopy (Benner, 2009) acetaldehyde glycoaldehyde relative abundances of molecules in space Small Molecule Precursors Found in space: • hydrogen cyanide (HCN) • acetlyene (HC CH) • formic acid (HCOOH) • formaldehyde (H2CO) • acetic acid (CH3COOH) • ammonia (NH3) • water Found in comets & meteorites: • amino acids • nucleobases • lipids • PAHs • water abundant on early Earth: hydrogen sulfide, CO, water, methane, salts, etc. ... but how? the Earth’s early atmosphere • once the Earth accreted, it formed a primary atmosphere • but it was soon able to evolve its own, secondary atmosphere through outgassing of its interior • in particular, the outgassing of H gradually but steadily 2 occurred (contemporary atmospheres of Venus, Earth, and Mars: Zubay Table 5-2) contemporary atmospheres of Venus, Earth, and Mars the Earth’s early atmosphere • three important molecules could then form in the early atmosphere: 1. water vapor (H2O) * 2. methane (CH4) 3. ammonia (NH3) • other gasses probably present: CO & N , 2 plus those that are currently outgassing: CO2, HCl, and H2S the Earth’s early atmosphere – the big question: oxidizing (e– poor) = “BAD” vs. reducing (e– rich) = “GOOD” the dominant view recently (e.g., Jim Kasting) has been that the primitive atmosphere was a weakly reducing mixture of CO2, N2, and H2O, combined with lesser amounts of CO and H2 the Earth’s early atmosphere – the big question: oxidizing vs. reducing any O2 made abiotically could have been lost from the atmosphere by reactions with: H2 (to give water) CO (to give carbonate) Si (to give silicates = glass) Fe(II) to give Fe(III) 4Fe(II)O + O2 banded iron 2Fe2(III)O3 four key reactions could have occurred in this type of atmosphere: abiotic formaldehyde 1. CO2 + 2H2 H2CO + H2O nitrogen photolysis 2. N2 + hν 2N abiotic methane 3. CO2 + 2H2O CH4 + 2O2 4. 2CH4 + 2N + hν 2HCN + 3H2 abiotic hydrogen cyanide H2CO and HCN were major players in future reactions!!! again, the OoL timing (4.0 +/- 0.1 bya) is bounded by two events: more recent boundary: oldest BIF dates to 3.85 bya more ancient boundary: severe meteoritic impacts still occurring once per 50,000 years at 4.2 bya some sources of small molecule precursors: H2, N2, CO, CH4, etc. • • • molecular hydrogen (H2) is not common in life, but may have been critical in the OoL for its roles in the formation of water and simple hydrocarbons gasses such as N2 and CO were very important, because they were the ultimate sources of nitrogen and reducible carbon, respectively hydrogen cyanide (HCN), acetylene (HCCH), and formaldehyde (H2C=O) are abundant in interstellar gasses; these molecules can provide reducing power (e–) for the OoL some sources of small molecule precursors: water • • • • • • water is the solvent of life today, 2/3 of the Earth’s surface is water water could have been abundant in significant (to the OoL) amounts on the early Earth as soon as 4.3 bya (Steve Mojzsis) water can be formed by the reduction of oxygen-containing compounds such as CO, but only at high temperatures or pressures, so this likely happened during the original accretion of the Earth after the Earth was formed, water was probably delivered by comets that impacted the Earth most of the Earth’s water likely had an extraterrestrial origin in space: 1. 3O2 + UV –> 2O3 2. O3 + 3H2 –> 3H2O the influence of the Solar System’s Big Brother Jupiter • • • • some of the volatiles on the early Earth were there because of the gaseous planets, Neptune, Uranus, Saturn, and particularly Jupiter the massive gravity of this planet helped to “clean up” the protoplanetary debris in the Solar System the debris either got ejected from the Solar System or condensed into the inner planets, where they could be delivered to Earth via meteorites carbonaceous chondrites: rich in carbon, 3% total organics, and 5% water with these few molecules, plus gasses, the larger components of life must have been made possible sources of energy for the OoL entry-level jobs Kelly Scientific Resources has an opening for an Extractions Technician at an environmental company in Tigard, OR. This position has the potential to become a temp to hire opportunity. The successful candidate will perform sample preparation duties including solvent extractions, solid phase extractions and clean-up techniques using EPA methods. Schedule is a late day shift starting around 10am till about 6pm. Kelly Scientific Resources has an opening for a Sample Receiving Technician at an Environmental Company in Tigard, OR. This position has the potential to become a temp to hire opportunity. Individual will be responsible for sample collection, shipping, receiving, technical data entry, and record keeping of samples received. Will also provide customer service to clients on the phone and in person. Kelly Scientific Resources has an opening for a Quality Assurance Technician at a Chemical Manufacturing company in Tacoma, Washington. The successful candidate will be responsible for analytical and physical testing of products produced within the production facility, monitoring of critical quality systems, reporting and documentation, as well as other general laboratory duties. Senior Lab Technician - Kelly Scientific Resources has an opening for a Chemical Lab Technician at a chemical manufacturing company in Hillsboro, Oregon on a temp-hire basis. If interested, please contact Kelly Scientific Resources directly at 503-245-4533 or send your resume to [email protected]. Kelly Scientific is a division of Kelly Services - an international, Fortune 500 Company with a reputation for excellence. Kelly Scientific is devoted to partnering with companies to provide scientific professionals with the best possible positions to meet their career goals. There is never a fee to our applicants or employees. For more information, visit www.kellyscientific.com. The Seven Challenges to a Prebiotic Chemist 1. The origin/source of the elements 2. The origin/source of small molecule precursors 3. The origin/source of monomers 4. The condensation problem 5. The self-replication problem 6. The chirality problem 7. The compartmentalization problem comets a comet is a small, “icy” Solar System body Darwin’s “Warm Little Pond” the primordial soup = the primordial ooze monomers O amino acids NH2 OH N N NH2 N O -O P O O OOH H O fatty acids H O H H H OH H OH nucleotides H OH HO HO H OH OH sugars H N the source of monomers e.g., amino acids H2 + NH3 + CH4 + H2O a “dead” bag of chemicals energy H2N – CH2 – COOH glycine, an amino acid early theories on the origins of life from a chemical evolution perspective • Darwin e.g., 1871 • JBS Haldane (1892–1964) • Alexandr Ivanovich Oparin (1894–1980) a “dead” bag of chemicals other, more complex chemicals JBS Haldane (British Geneticist) • Haldane thought much about prebiotic chemistry, but, as a geneticist, did few actual experiments on the topic • In 1923, gave a talk at Cambridge on the possibility of hydrogen-generating windmills as an alternative to coal fuel • In 1925, developed the Briggs–Haldane derivation of the Michaelis-Menten enzyme kinetic equation • In 1929, wrote an article for the Rationalist Annual called “The Origin of Life” • may have coined the phrase “prebiotic soup” JBS Haldane The Earth’s earliest atmosphere would have been devoid of molecular oxygen, and rather, comprised of ammonia and carbon dioxide. Without O2, there would be no O3 to protect the Earth from ultraviolet radiation, which could have provided energy for the polymerization of small molecules into proteins Alexandr Ivanovich Oparin (Russian Biochemist) Oparin • Oparin postulated a long chemical evolution as a necessary preamble to the emergence of life • He devised a sequence of plausible reactions, and then actually did some experimentation to test his ideas • Was perhaps the first to seriously consider the abiotic origins of cell-like structures Oparin • Wrote a seminal book on the topic in Russian in 1924 • He was really the first to consider the incoming data on the formation and composition of the Sun and the planets • In the early 1930’s it was possible to study the Sun’s elemental make-up and to observe the atmospheric compositions of nearby planets, especially Venus English edition, first published in 1938 Oparin’s Chemical Evolution • His first conclusion: carbon made its first appearance on the Earth not in the oxidized form of CO2 but in the reduced form of hydrocarbons • He believed the Earth’s earliest atmosphere was strongly reducing • Was influenced by experiments of other Russians that showed that iron carbides could react with hot water to generate hydrocarbons: 3FemCn + 4mH2O mFe3O4 + C3nH8m e.g., m = n = 1: 3FeC + 4H2O Fe3O4 + C3H8 iron in reduced state (Fe(II)) is converted to a mixed oxidation state during the reduction of carbide to propane Oparin’s ideas on the early atmosphere • Was concerned about the source of nitrogen, because of its important role in proteins • He didn’t think the early atmosphere contained much O2 or N2 • Thus he proposed that nitrogen first became trapped in the Earth’s core at high temperatures by the formation of metal nitrides, then released as ammonia upon oxidation by water vapor: Δ Mg3N2; 2Al + N2 1. 3Mg + N2 Fe(OH)3 + NH3 2. FeN + 3H2O Δ Al2N2; 2Fe + N2 Δ 2FeN another possibility: the Haber production of ammonia, occurring in the upper portions of the Earth’s crust Oparin’s pathway from simple hydrocarbons to more complex biologically relevant molecules aldehydes (e.g., acetaldehyde) could have been produced by the hydration of acetylene: CH CH + H2O CH3CHO two acetaldehyde molecules could have condensed by an aldol condensation reaction to give an alcohol: 2CH3CHO CH3CHOHCH2CH2OH a succession of such condensations could have led to glucose, a polyol: the aldol condensation reaction two aldehydes condense to form a more complex alcohol: 1. tautomerization of an aldehyde to an enol or enolate (base catalyzed) 2. nucleophilic attack of the enol on the carbonyl center of another aldehyde to give an addition product 3. re-protonation to give the β-hydroxy aldehyde Geoffrey Zubay: “The synthesis of sugars in the prebiotic world is likely to have started with formaldehyde” the aldol condensation reaction later, we will see the importance of this type of process in driving the “formose reaction” nCH2O (CH2O)n {the fixation of formaldehyde into carbohydrates} Oparin’s realized the problem of concentrations! • prebiotic chemistry has an intrinsic problem in that a series of reactions with <100% yields mandates lower and lower probabilities of products with each additional step • if each step occurs in low yield, or if the concentrations of precursors is low, then the overall yield is in danger of being so small as to be negligible • the high concentrations of water on the early Earth would have diluted reactants, diffused away products, AND inhibited condensation reactions • Oparin proposed that simple cell-like structures called coacervates were needed at or near the origins of life to deal with these issues Oparin’s coacervates 1 – 500 μm in diameter Coacervates, which are polymer-rich collodial droplets, were studied in the Moscow laboratory of Oparin because of their conjectural resemblance to prebiological entities. These coacervates are droplets formed in an aqueous solution of protamine and polyadenylic acid. Oparin found that droplets survive longer if they can carry out polymerization reactions inside. The Seven Challenges to a Prebiotic Chemist 1. The origin/source of the elements 2. The origin/source of small molecule precursors 3. The origin/source of monomers 4. The condensation problem 5. The self-replication problem 6. The chirality problem 7. The compartmentalization problem the source of monomers amino acids H2 + NH3 + CH4 + H2O a “dead” bag of chemicals energy H2N – CH2 – COOH glycine, an amino acid The Miller-Urey spark-discharge experiments the source of monomers amino acids glycine, alanine, aspartic acid, etc. the Miller-Urey spark-discharge experiments (1953-2000) The original Miller-Urey Experiment (1952) CH4 (20 torr) + NH3 (20 torr) + H2 (10 torr) + H2O (vapor) 500 mL flask: water (“ocean”) + 2 L flask: gas (“atmosphere”) 2000 V spark; one-week incubation time Miller (1953) Science 111:528–529. The original Miller-Urey Experiment (1952) paper chromatography CH4 + NH3 + H2O + H2 + energy : glycine > α-alanine > α-amino-n-butyric acid > β-alanine > glutamic acid > aspartic acid Results from the original Miller-Urey Experiment (1952) overall, about 15% of the carbon in methane is converted to intermediate-sized molecules by this technique = Table 4.2 in P&G subsequent Miller-Urey experiments (1953–) varied the input gasses & concentrations all the way from strongly reducing (best yields) to mildly oxidizing (poorer yields) varied flask configurations and gas pressure varied energy source (e– vs. UV vs. heat, etc.) & time subsequent Miller-Urey experiments (1953–) proteinaceaous amino acids, their isomers, and other amino acids that are formed; total AA yield = 1.90% = Table 4.3 in P&G intermediates in Miller-Urey experiments the appearance and then disappearance of HCN and aldehydes reveals that they are key intermediates = P&G Fig. 4.4 2000V: produces free radicals to drive production of intermediates variant Strecker synthesis of amino acids and hydroxy acids 0. the production of aldehydes and HCN via free-radical chemistry from simple gaseous starting materials, for example: a) CH4 + H2O [CH4 + e–* b) 2CH4 + N2 [N2 + e–* H2CO + H2 CH3 + H+] 2HCN + 3H2 2N ] 1. the production of a cyanoamine: RCH=O + NH3 + HC N RCHNH2C N 2. the hydration of the cyanoamine to give an amino acid: NH2 RCHNH2C N +2H2O R–C–COOH H the classic Strecker synthesis of amino acids the Strecker synthesis of amino acids and hydroxy acids 1. the addition of ammonia to an aldehyde to give an imine: 2. the addition of cyanide to the imine to give a cyanoamine (aminonitrile): 3. hydrolysis of the cyanoamine to give an amino acid: 2´ & 3´. the addition of cyanide to the aldehyde directly and then hydrolysis gives a hydroxy acid instead: = Fig. 4.5 in P&G cyano compounds of prebiological interest • HC N (hydrogen cyanide): basic precursor to almost all biological monomers; formed from CH4 and NH3 • N C–NH2 (cyanamide): activator for peptide condensation • N C–C CH (cyanoacetylene): formed from CH4 and N2; used in pyrimidine abiosynthesis; used in Asp and Asn abiosynthesis • N C–CH=NH (iminoacetonitrile): HCN dimer; used in purine abiosynthesis • R–CH2(NH2)–C N (aminonitriles) & R–CH2(OH)–C (hyrdoxynitriles): used in amino acid abiosynthesis N the Strecker synthesis should produce a racemic mixture amino acids found in the Miller experiments are indeed racemic; amino acids found in meteorites have some ee; amino acids in proteins are all L Collision in the asteroid belt! Potential meteorites! courtesy of Dave Deamer courtesy of Dave Deamer September 28, 1969 Murchison, Australia 5! courtesy of Dave Deamer the amino acids in the Miller-Urey syntheses match those found in meteorites (such as the Murchison) rather well meteorites contain detectable amounts of many amino acids, especially glycine, alanine, and αamino-n-butyric acid, along with a range of hydroxy acids the Miller-Urey experiments have produced at least 17 of the 20 or so proteinaceaous amino acids some require subsequent modifications the three aromatics, Tyr, Trp, and Phe require an alternative synthetic route Miller has proposed an abiotic route to histidine that mimics the biosynthetic route erythrose would come from the formose reaction (coming soon!) Miller’s experiment generated instant media attention “Milk, meat, albumen, bacteria, viruses, lungs, hearts – all are proteins. Wherever there is life there is protein” stated the New York Times in its May 15, 1953 issue. “Protein is of fairly recent origin, considering the hot state of the earth in the beginning. How the proteins and therefore life originated has puzzled biologists and chemists for generations. Accepting the speculations of the Russian scientist A. I. Oparin of the Soviet Academy of Science, Prof. Harold C. Urey assumes that in its early days the earth had an atmosphere of methane (marsh gas), ammonia and water. Oparin suggested highly complex but plausible mechanisms for the synthesis of protein and hence of life from such compounds. In a communication which he publishes in Science, one of Professor Urey’s students, Stanley L. Miller, describes how he tested this hypothesis”, continued the New York Times, “A laboratory earth was created. It did not in the least resemble the pristine earth of two or three billion years ago; for it was made of glass. Water boiled in a flask so that the steam mixed with Oparin’s gases. This atmosphere was electrified by what engineers call a corona discharge. Miller hoped that in this way he would cause the gases in his artificial atmosphere to form compounds that might be precursors of amino acids, these amino acids being the He actually synthesized some amino acids and thus made chemical history by taking the first step that may lead a century or so hence to the creation of something chemically like beefsteak or white of egg. bricks out of which multifarious kinds of protein are built. Miller is elated, and so is Professor Urey, his mentor.” The Seven Challenges to a Prebiotic Chemist 1. The origin/source of the elements 2. The origin/source of small molecule precursors 3. The origin/source of monomers 4. The condensation problem 5. The self-replication problem 6. The chirality problem 7. The compartmentalization problem cyano compounds of prebiological interest • HC N (hydrogen cyanide): basic precursor to almost all biological monomers; formed from CH4 and NH3 • N C–NH2 (cyanamide): activator for peptide condensation • N C–C CH (cyanoacetylene): formed from CH4 and N2; used in pyrimidine abiosynthesis; used in Asp and Asn abiosynthesis • N C–CH=NH (iminoacetonitrile): HCN dimer; used in purine abiosynthesis • R–CH2(NH2)–C N (aminonitriles) & R–CH2(OH)–C (hyrdoxynitriles): used in amino acid abiosynthesis N The RNA World • a proposed period of time when RNA (or something like RNA) was responsible for all metabolic and information-transmission processes • RNA has both a genotype AND a phenotype (Cech, Altman: catalytic RNA ... Nobel Prize, 1989) • Catalytic RNA = ribozymes (9 classes) • The ribosome is a ribozyme The RNA World... ...needs ribose, nucleobases, and phosphates The Source of Monomers ribose sugars O HO OH OH OH ribose requires 5 carbons, C-O bonds, and correct stereochemistry two acetaldehyde molecules could have condensed by an aldol condensation reaction to give an alcohol: 2CH3CHO CH3CHOHCH2CH2OH a succession of such condensations could have led to glucose, a polyol: The Source of Monomers ribose sugars glycoaldehyde formaldehyde DL-glyceraldehyde ribose The formose reaction (autocatalytic) the formose reaction Butlerov (1860): formaldehyde + water + calcium hydroxide + heat gives a mixture of sugars O HO OH OH formaldehyde is used to make glycoaldehyde, trioses, and tetroses; pentoses such as ribose are made by the condensation of glycoaldehyde and a triose OH the formose reaction optimal: high pH, calcium hydroxide, 55˚C, 1-2% aqueous formaldehyde • • • • The formose reaction exploits the natural nucleophilicity of the enediolate of glycoaldehyde and the natural electrophilicity of formaldehyde. The calcium ion stabilizes the enediolate of glycoladehdye. This species reacts as a nucleophile with formaldehyde (acting as an electrophile) to give glyceraldehyde. Reaction of glyceraldehyde with a 2nd equivalent of the enediolate generates a pentose sugar (ribose, arabinose, xylose, or lyxose) The formose reaction is autocatalytic DL-glyceraldehyde glycoaldehyde tetrose glycoaldehyde is the autocatalytic reagent: it is both the product of the condensing of two formaledhyde molecules AND a catalyst for this condensation The formose reaction is autocatalytic C3: DL-glyceraldehyde C2: glycoaldehyde glycoaldehyde is the autocatalytic reagent: it is both the product of the condensing of two formaledhyde molecules AND a catalyst for this condensation The formose reaction is autocatalytic glycoaldehyde = Fig. 4.7 P&G the glycoaldehyde cycle ribose is but one of many possible 5-carbon sugars: 3C 4C 5C 6C then the straight-chain form must cyclize: (6C example) The formose reaction produces a dizzying array of products ribose GC Decker, Schweer, & Pohlmann (1982) J. Chromatogr. 244: 281–291. The formose reaction can make ribose, but the yield is poor (<1%) and MANY other products arise Possible solutions: • phosphorylating the glycoaldehyde (Eschenmoser, 1990) • using lead salts and mildly basic conditions (Zubay, 1998) • boron complexation (Benner, 2004) • membranes can be selectively permable (Szostak, 2005) • silicate complexes (Lambert, 2010) • alternative backbones: PNA, TNA, etc. Albert Eschenmoser: use phosphate! Using phosphorylated glycoaldehyde not only give you phosphorylated sugars, but it also greatly biases products towards ribose: Geoff Zubay: use lead! Lead (II) ions can increase the yields of aldopentoses from formaldehyde by over 20-fold Zubay, 1998 the power of lead (II) is a result of its high affinity for cis-hydroxyls and its very low pKa value (the pKa of hydrated lead (II) ions is about 7.7) Steve Benner: use borate! Borate ions can stabilize glyceraldehydes, preventing them from acting as nucleophiles and thus stemming out-of-control polymerization O HO HO HH O H ulexite NaCaB5O9•8H2O OH O O B O glycoaldehyde + DL-glyceraldehyde Ca(OH)12 boron mineral O pentoses as majority Ricardo, Carrigan, Olcott, & Benner (2004) Science 303, 196 Jack Szosak: use cell membranes! Sacerdote and Szostak (2005). Proc. Natl. Acad. Sci. USA,102:17–22. using certain phospholipid membranes in artificial cells results in a greatly increased permeability to ribose vs. other pentoses and sugars Joseph Lambert: use silicates! aqueous sodium silicate can select for sugars with a specific stereochemistry Lambert et al. (2010). Science,327:984–986. maybe ribose came later, and simpler backbones came first: GNA: glycerol-derived acyclonucleic acid TNA: threose nucleic acid p-RNA: pyranose RNA maybe ribose came later, and simpler backbones came first: TNA PNA: peptide nucleic acid GNA p-RNA Joyce (2004) The Seven Challenges to a Prebiotic Chemist 1. The origin/source of the elements 2. The origin/source of small molecule precursors 3. The origin/source of monomers 4. The condensation problem 5. The self-replication problem 6. The chirality problem 7. The compartmentalization problem The RNA World... ...needs ribose AND nucleobases, AND phosphates conventional wisdom: 1a. make nucleobase 1b. make ribose (e.g., formose rxn) 1c. find phosphate source 2. add base to sugar 3. add phosphate the source of monomers nucleobases NH2 hydrogen cyanide (HCN) 5 H C N present in interstellar medium adenine N H N H 15 atoms & 50 electrons: 5 C-H bonds 5 C-N bonds read P&G’s discussion of HCN on the Earth (pp. 95-97) N recombination N H 15 atoms & 50 electrons: 2 C-H bonds 9 C-N bonds 3 N-H bonds 1 C-C bond present in living systems the Oró HCN polymerization experiments (1961-) the mechanism of Oró HCN polymerization HCN 1. 2. 3. 4. 5. dimerization of HCN trimerization to aminomaleonitrile tetramerization to DAMN UV-induced isomerization final HCN addition and ring closure adenine “We come from stardust and stardust we will become. We must be humble, because life comes from very simple molecules. We must be supportive, because we have a common origin. We have to be cooperative, since from the Moon the Earth is seen as a speck lost in the vastness of space, where the boundaries between people and the color of their skin cannot be distinguished.” Joan Oró (1976) the mechanism of Oró HCN polymerization optimum rate at pH 9.2 (pKa of HCN) = P&G Fig. 4.9 1. 2. 3. 4. 5. dimerization of HCN trimerization to aminomaleonitrile tetramerization to DAMN UV-induced isomerization final HCN addition and ring closure iminoacetonitrile the mechanism of Oró HCN polymerization = P&G Fig. 4.9 1. 2. 3. 4. 5. dimerization of HCN trimerization to aminomaleonitrile tetramerization to DAMN UV-induced isomerization final HCN addition and ring closure the mechanism of Oró HCN polymerization 1. 2. 3. 4. 5. dimerization of HCN trimerization to aminomaleonitrile tetramerization to DAMN UV-induced isomerization final HCN addition and ring closure Zubay: last HCN addition may come after a formylation instead, akin to purine biosynthesis Adenine Guanine AICA equivalent biosynthesis of purines HCN polymerization (courtesy of Tim Riley) other purines pyrimidines -- more difficult Various pyrimidines can be formed using UV light in ammonia-rich ices Nuevo et al. (2012) Astrobiology 12: 295–314 attaching base to sugar... O N N O -O P O NH N O OH H H OH H OH IMP Leslie Orgel: hypoxanthine + D-ribose + Mg2+ gives β-inosine under dehydrating conditions (low yield) this reaction does not work for the pyrimidines! The Source of Monomers phosphates Possible sources of phosphates: • fluorapatite in Earth’s crust: Ca10(PO4)6F2 • schreibersite in iron meteorites: (Fe, Ni)3P • alkyl phosphonic acids in meteorites: R–H2PO3 Nearly all phosphorus in the Earth’s crust is in the form of orthophosphate, which has low reactivity toward organic compounds, and thus phosphate minerals are not good bets for the abiotic P source. phosphorus compounds phosphates from more reduced forms of P schreibersite is a rare iron-nickel phosphide mineral, but is common in iron-nickel meteorites There is evidence that schreibersite, when dissolved in water, can form pyrophosphate, which can phosphorylate sugars (Matt Pasek, U. Arizona) evolution of molecular hydrogen after soaking of Fe3P in water, indicating the production of phosphates Pasek & Lauretta (2005) Astrobiology 5: 515–535. The Source of Monomers making a complete nucleotide RNA-catalyzed nucleotide assembly? Joyce (2002) example: nucleotide synthetase ribozyme Unrau & Bartel (1998) Nature 395, 260-263 The Source of Monomers making a complete nucleotide A difficult task! Could RNA have been a “biotic invention”? {Anastasi et al. (2007)} a new strategy?!? cyanamide 8 + cyanoacetylene 7 + glycoaldehyde 10 + glyceraldehyde 9 + inorganic phosphate*** arabanose amino-oxazoline 12 β-D-ribocytidine 2´,3´ phosphate (oh yeah!) Powner, Gerland, and Sutherland (2009) Nature 459, 239–242 “the prebiotic synthesis of activated pyrimidine nucleotides should be viewed as predisposed” Powner et al. (2009) Nature 459, 239–242 a three-fer! movie “Although inorganic phosphate is only incorporated into the nucleotides at a late stage of the sequence, its presence from the start is essential as it controls three reactions in the earlier stages by acting as a general acid/base catalyst, a nucleophilic catalyst, a pH buffer and a chemical buffer.” 1M phosphate buffer, pH 7, 40˚C, o/n Powner, Gerland, and Sutherland (2009) Nature 459, 239–242 The Seven Challenges to a Prebiotic Chemist 1. The origin/source of the elements 2. The origin/source of small molecule precursors 3. The origin/source of monomers 4. The condensation problem 5. The self-replication problem 6. The chirality problem 7. The compartmentalization problem Condensation • polymerizing monomers with the liberation of water ... in water! O H2N CH C CH3 OH + H2N CH C CH2 OH Ala O O Ser OH H2N CH CH3 C O H N CH C OH CH2 OH + H2O activating groups and/or condensing agents were probably important for prebiotic chemistry • cyanamide • imidizole • thioesters • phosphoanhydrides (used in biology today!) possible mechanisms of amino-acid condensation • heating of dry amino acids to get “proteinoids” (Fox) • thermal condensation on clay (Chang, Ferris) • cyanamide-mediated synthesis (Oro) Sydney Fox’s proteinoids (debunked) Nature 129: 1221–1223 (1959) Thermal condensation on clay Science 201: 67–69 (1978) Lahav, N., White, D., Chang, S. Cyanamide-mediated polymerization (draw mechanism on whiteboard) J.Mol. Evol. 17: 285–294 (1981) The RNA World... ...needs ribose, nucleobases, and phosphates ... and chains! 5´-GUGCCUUGCGCCGGGAAACCAC...-3´ RNA structure Azoarcus ribozyme (205 nt) Adams et al. (2004) Nature 430, 45-50. The Catalytic Repertoire of RNA Chen, Li, & Ellington (2007) The Source of Polymers NH2 N N O -O P O N N O OH '5 H H OH H OH • activation is needed: triphosphate, • linakage geometry is important • templating can help A A A 3' imidizole, etc. contemporary polymerases in-line nucleophilic attack Figure 30-10 Schematic diagram for the nucleotidyl transferase mechanism of DNA polymerases. abiotic RNA polymerization 1. high-energy condensing agents 1.1. amino acid adenylates 1.2. imidizolides 1.3. water-soluble carbodiimides 1.4. purines and pyrimidines 2. catalytic action 2.1. inorganic ions 2.2. clays 2.3. oligonucleotide templates 2.4. ribozymes 2.5. lipids amino acid adenylates NH2 N NH2 O O N O P O N N O OH H H OH H OH nucleotides have been proposed to condense amino acids, so can the reverse be true: AA used to condense nt’s? imidazolides NH2 N –HO: N O N N P O R = H or CH3 N O OR N H H H OH H OH ImpA see P&G, Fig. 4.16 far more active as condensing agents, because the imidizole moiety is a good leaving group that allows for a successful attack of hydroxyl groups on a phosphorus center water-soluble carbodiimides R1–N=C=N–R2 example: EDC = 1-ethyl-3(3-dimethylaminopropyl)carbodiimide phosphoramidite purines and pyrimidines NH2 N N O N N P O N N NH2 O OH H H OH H OH N N O 4-dimethylaminopyridinium-AMP N N P O N N O OH2N N H H H OH H OH N H3C adenosine-5´-phophoro-1-methyladeninium purine- and pyrimidine-like molecules are attached to the 5´ phosphate and serve as good leaving groups catalysts for RNA condensation: points to consider 1. template-directed vs. non-template directed 2. all 3´-5´ linkages vs. mixture of 3´-5´ and 2´-5´ 3. autocatalytic vs. non-autocatalytic catalysts for RNA condensation ions: inorganic cations such as Zn(II), Pb(II), and UO2(II) have been demonstrated empirically to speed up RNA polymerization in the lab clays: montmorillonite clays have been demonstrated empirically to speed up RNA polymerization in the lab templates: pre-existing polymer templates have been demonstrated empirically to speed up RNA polymerization in the lab example study #1: Lohrmann, Bridson, & Orgel (1980) Science 208: 1464–1465 HPLC elution profiles of products from the template-directed selfcondensation of ImpG in the presence of (a) 0.01 M Pb(II) or (b) 0.04 M Zn(II). 0.02 M ImpG, 0.04 M poly(C), 0.4 M NaNO3, 0.5 M Mg(NO3)2, 12 days, 0˚C, pH 7 example study #2: Sievers & von Kiedrowski (1994) Nature 369: 221–224 cross-catalytic schemes: auto-catalytic schemes: example study #2: Sievers & von Kiedrowski (1994) Nature 369: 221–224 A = CCG B = CGG Self-complementary autocatalysis has been previously demonstrated, but nucleic acid replication utilizes complementary strands, which can replicate via cross-catalysis example study #2: Sievers & von Kiedrowski (1994) Nature 369: 221–224 example study #2: Sievers & von Kiedrowski (1994) Nature 369: 221–224 AB BA, AA, and BB the addition of a particular product enhanced the rate of synthesis of that one product only example study #3: Ferris et al. (1996) Nature 381: 59–61 Clays to the Rescue? • some aluminosilicate sheets have positive charges AND a correct spacing to fit activated nucleotides into pockets • daily “feeding” of montmorillonite clay & a primer with activated nucleotides leads to polymerization without a template! Ferris et al. (1996) Nature 381: 59–61 Jim Ferris: daily “feeding” of nucleotides to clay results in RNA chains! longer RNA chains shorter RNA chains the correct linkage and stereochemistry can be achieved Joshi, Aldersley, Zagorevskii, & Ferris (2012) Nucleosides, Nucleotides, & Nucleic Acids, in press Clays: layers of ions example: Montmorillonite Jim Ferris: “A key to our eventual success was the discovery that montmorillonite-catalyzed reactions of nucleotides work best when we convert clays to forms with a single kind of interlayer cation—a procedure that avoids reactions or inhibition due to the metal ions bound in the interlayers of the naturally occurring montmorillonite (Banin 1973). We accomplished this conversion either by treatment of the montmorillonite with excess salts of the cation (saturation procedure) or by conversion to the acid form by acid treatment and then back titration of the hydrogen form of the clay with the desired cation. We observed that when the alkali and alkaline earth metal ions (with the exception of Mg) are the exchangeable cations, catalytically active clays are obtained.” The Seven Challenges to a Prebiotic Chemist 1. The origin/source of the elements 2. The origin/source of small molecule precursors 3. The origin/source of monomers 4. The condensation problem 5. The self-replication problem 6. The chirality problem 7. The compartmentalization problem RNA making RNA: self-replication – – + + • how do you transfer information from one molecule to another? • balance between fidelity (for information maintenance) and errors (for evolution) naturally existing catalytic RNAs group I introns (nucleotidyl transfer / transesterification) group II introns (nucleotidyl transfer / transesterification) RNase P (phosphodiester hydrolysis) ribosome (peptidyl transfer) hammerhead ribozymes (transesterification) hairpin ribozymes (transesterification) HDV ribozymes (transesterification) neurospora VS (transesterification) riboswitch ribozyme (transesterification) RNA-directed catalysis in natural ribozymes phosphoester bond cleavage (hydrolysis) trans-esterification 2´ -OH attack trans-esterification 3´ -OH attack self-cleaving ribozymes & reversibility this molecule should look familiar! group I intron ribozyme Azoarcus ribozyme (205 nt) Adams et al. (2004) Nature 430, 45-50. in vitro selection (test-tube evolution) phenotype assay selection scheme Joyce (2007) ACIE The Catalytic Repertoire of RNA Chen, Li, & Ellington (2007) RNA making RNA: self-replication the “holy grail” of prebiotic chemistry: discovery of an RNA autoreplicase a significant advance towards this goal: the Bartel ligase ribozyme Johnston et al. (2001) Science 292, 883-896. Zaher & Unrau (2007) RNA 13, 1017-1026. Wochner et al. (2011) Science 332, 209-212. RNA making RNA: the Bartel/Unrau replicase ribozyme a 190-nt ribozyme that can polymerize up to 95 nt : polymerase chemistry: class I ligase ribozyme NNNN–OH + pppN b201 ligase (Bartel & Szostak, 1993) In vitro selection of the original replicase ribozyme (2001) class I ligase ribozyme primer (orange) + template (red) replicase-14 Johnston et al. (2001) Science 292, 883-896. template extension by replicase-14 Johnston et al. (2001) Science 292, 883-896. fidelity of replicase-14 Johnston et al. (2001) Science 292, 883-896. In vitro selection of an improved replicase ribozyme (2007) replicase-14 in vitro selection Zaher & Unrau (2007) RNA 13, 1017-1026. water-in-oil emulsions In vitro selection of an improved replicase ribozyme (2007) replicase-20 up to 20 nt, with 3–4fold more accuracy Zaher & Unrau (2007) RNA 13, 1017-1026. In vitro selection of an even more improved replicase ribozyme (2011) replicase-95 the tC19Z ribozyme (replicase-95) can polymerize up to 95 nt! 95/187 = 50% Wochner et al. (2011) Science 332, 209-212. up to 95 nt, but only certain templates Eigen’s error threshold Q: how accurate must a replicase be to maintain information in a population of (RNAs)? A: the length is limited by, ν < –ln σm / ln q where we are considering a selfreplicating RNA formed by ν condensation reactions, each having a mean fidelity q, where σm is the relative selective “superiority” of the advantageous individual compared to the remainder of the population Eigen’s error threshold Roughly, to maintain information, the length of a self-replicating RNA must be less than the inverse of its error rate replicase-14: fidelity = 0.967, thus μ = 1 – 0.967 = 0.033 νmax = 1/0.033 = 30 nt replicase-20 μ = 0.011 νmax = 1/0.011 = 92 nt The Origin of Chirality “asymmetry is a hallmark of life” modern biology: beta-D-ribonucleotides & L-amino acids it’s not clear how these were selected out of a racemic mixture; moreover there is enantiomeric cross-inhibition life is chiral; this is a “biosignature” Earth life: L-amino acids and D-nucleotides Text abiotic material is achiral or racemic the origin of chirality “asymmetry is a hallmark of life” modern biology: beta-D-ribonucleotides & L-amino acids it’s not clear how these were selected out of a racemic mixture, but possible solutions include: assistance from a chiral surface (e.g., quartz), differential precipitation or solvation, slightly different energies of the two enantiomers chiral symmetry breaking by CPL enantiomeric cross inhibition could have lead to the origin of chiral synthesis? Zubay Fig. 14-10; Joyce et al. (1987) The Seven Challenges to a Prebiotic Chemist 1. The origin/source of the elements 2. The origin/source of small molecule precursors 3. The origin/source of monomers 4. The condensation problem 5. The self-replication problem 6. The chirality problem metabolism FIRST? 7. The compartmentalization problem Metabolism-first Theories the notion that without energy-generating mechanisms in place, life could not have originated • Christian De Duve’s “Thioester World” • Gunter Wächtershäuser’s “Pyrite World” • George Cody’s “Nickel-iron-sulfur CO-transfer World” the thioester world De Duve has proposed thioesters as a key molecule to allow the build-up of larger molecules R1 O S C R2 De Duve: “without additional help of both catalytic and energetic nature, the prebiotic broth would have remained sterile” origin of thioesters R1 SH thiol + energy O R2 C OH carboxylic acid H+ R1 O S C R2 thioester e.g., H2S would have been abundant on the prebiotic Earth, and simple carboxylic acids could have derived from MillerUrey type reactions origin of thioesters in a hot acidic environment the thiol group in thioesters is quite transferable R1 O S C R2 thioester + 2H+ + 2e– R1 SH reducing power thiol + thioester-dependent reductions R2 O C H aldehyde the thiol group in thioesters is quite transferable R1 O S C R2 thioester O + HO P O OH inorganic phosphate R1 SH thiol + O O R2 C O P O OH acyl phosphate thioester-dependent phosphorylations the thiol group in thioesters is quite transferable O R' S C R1 + O R' O R' S C R2 S C R2 dimer R1 + R' SH thiol thioester carriers thioester-dependent catalytic production of multimers De Duve: thioesters were used for general activation and sequential group transfer from “Blueprint for a Cell” (1991) the pyrite world hydrogen sulfide, in combination with the two redox states of iron, could have provided the functional precursors of all extant biochemicals FeS + H2S iron hydrogen sulfide sulfide 2H+ + 2e– + FeS2 reducing power pyrite Wächtershäuser views metabolism as primitive, and “inventing” a genetic structure later to maintain itself the pyrite world at deep-sea hydrothermal vents are large columns of percipitated salts, commonly including pyrite (FeS2) Wächtershäuser’s chemoautotrophic origins of life “local chemoautotrophic origin of life in hot volcanic exhalations by synthetic autocatalytic domino reactions of low molecular organic constituents on mineral surfaces of transition metal sulfides,” pyrite-pulled metabolism FeS + H2S CO2 + H2 FeS + CO2 + H2S H2 + FeS2 HCOOH HCOOH + FeS2 coupling an unfavorable reaction (the reduction of CO2) with a favorable one (pyrite production from pyrrhotite) could have led to the prebiotic fixation of carbon carbon monoxide can be converted to acetic acid first, iron sulfide is carbonylated: 2FeS + 6CO + 2R-SH 2S0 + H2 + Fe2(RS)2(CO)6 then the carbonylated Fe-S intermediate can be “desulfurized” to generate acetic acid and pyruvate: Fe2(RS)2(CO)6 CH3COOH + CH3-CO-COOH amino acids can polymerize upon activation by CO on FeS/NiS solid surfaces Huber & Wachtershauser (1998) Science 281: 670–672. pyrite-pulled metabolism (draw scheme on whiteboard) FeS/H2S might be able to reduce the relatively oxidized (electron-poor) hydrocarbons such as acetylene that are present in the interstellar dust the TCA cycle: at the root of anabolism “all extant organisms oxidize chemical fuels” to generate reducing power for metabolism the cycle traces both the number of carbons and their relative oxidation states generates reducing power the reductive TCA cycle: in biology, this is catalyzed by the acetyl-CoA synthase enzyme complex ... using an Fe-S cluser carbon fixation ... performed by protein enzymes containing Fe-S clusters! reducing power used to fix inorganic carbon the acetyl CoA pathway portion = the direct formation of acetate from CO2 or CO the origins of the acetyl-coA cycle: Cody’s suggestion an attractive feature of the pyrite world is the notion of life developing on a mineral surface (2D), aided by catalysts such as FeS2 also, FeS2 is similar to iron-sulfur clusters in the core of key enzymes in the TCA cycle! the origins of the acetyl-coA cycle: Cody’s suggestion the reactions taking place within the acetylCoA synthase enzyme require an Fe-S cluster at the core protometabolic carbon fixation Fe-S clusters can reduce CO to a transferable methyl group The Seven Challenges to a Prebiotic Chemist 1. The origin/source of the elements 2. The origin/source of small molecule precursors 3. The origin/source of monomers 4. The condensation problem 5. The self-replication problem 6. The chirality problem 7. The compartmentalization problem the three “stages” in the evolution of life 1. chemical evolution 2. self-organization 3. biological evolution the origin of cells “linking genotype with phenotype” compartmentalization would offer life enormous advantages • keeping water concentrations low • keeping local concentrations of solutes high • dividing protocell into distinct compartments • creating gradients • allowing genotypes to harvest “the fruits of their labor” protocell theories • Oparin’s coacervates • Fox’s proteinoid microspheres • liposomes (Deamer, Szostak, etc.) Oparin’s coacervates 1 – 500 μM in diameter Coacervates, which are polymer-rich collodial droplets, were studied in the Moscow laboratory of Oparin because of their conjectural resemblance to prebiological entities. These coacervates are droplets formed in an aqueous solution of protamine and polyadenylic acid. Oparin found that droplets survive longer if they can carry out polymerization reactions inside. Oparin’s coacervates (artificial!) Coacervates can be made by mixing: 1. proteins and carbohydrates (e.g., histones + gum arabic) 2. proteins and other proteins (e.g., histones + albumin) 3. proteins and nucleic acids (e.g., histones + RNA or DNA) Coacervates can encapsulate enzymes which are functional: phosphorylase Sydney Fox’s proteinoids (debunked) Nature 129: 1221–1223 (1959) liposomes when phospholipids are dissolved in water and then sonicated, the molecules tend to arrange themselves to form liposomes: closed, self-sealing, solvent-filled vesicles that are bounded by only a single layer liposomes lipids can self-organize to produce small droplets (micelles) or more complex structures containing bilayers liposomes monolayers can be converted to bilayers by agitation phospholipids lipids are a condensation of one or more fatty acids onto a poly-alcohol (a polyol) glycerol is a tri-ol that commonly serves as a foundation for the addition of hydrophic head groups such as phosphate and hydrophobic tail groups such as fatty acids phospholipids modern example fatty acids long aliphatic hydrocarbon chains, with or without unsaturated C–C bonds amphipathic molecules “self-assemble” lipid synthesis – today 1. make fatty acid side chains 2. esterify side chains to polyol lipid synthesis – abiotic 1. make side chains 2. esterify side chains to polyol Fischer/Tropsch reaction C + H2O Fe, Ni CnH2n+2 Δ addition of successive CO units lipid synthesis – abiotic 1. make side chains 2. esterify side chains Wachtershauser’s proposal CH2O FeS2 / H2S Δ (100˚C, pH7) CH2 = CH2 lipid synthesis – abiotic 1. make side chains 2. esterify side chains to polyol Art Weber’s hypothesis • uses glycoaldehyde as an acyl carrier • is a cycle of condensation, dehydration, and isomerizations • does not require ATP input • can be catalyzed by metal ions abiotic lipid synthesis tied to abiotic ribose synthesis through glyceraldehyde? lipid synthesis – abiotic 1. make side chains 2. esterify side chains to polyol glycerol + FA + phosphate, then ... ...dehydration & rehydration Artificial Cell Research Dave Deamer & Jack Szostak • synthetic cells can encapsulate active enzymes: Chakrabarti et al. (1994). J. Mol. Evol. 39:555–559. • synthetic cell membranes can select for ribose: Sacerdote and Szostak (2005). Proc. Natl. Acad. Sci. USA102:6004–6008. Dave Deamer: liposome research Dave Deamer: liposome research the chemiosmotic potential of membranes could have driven abiotic syntheses encapsulation of polynucleotide phosphorylase (PNP) Chakrabarti AC, Breaker RB, Joyce GF, and Deamer DW (1994). Production of RNA by a polymerase protein encapsulated within phospholipid vesicles. J. Mol. Evol. 39:555–559. Dave Deamer: liposome research phosporylase Chakrabarti et al. (1994). J. Mol. Evol. 39:555–559. methods 1.the lipid DMPC (dimyrisoyl phosphatidyl choline) was sonicated in water 2.dry PNPase added & mixture dried under N2 gas 3.rehydration in buffer 4.extrusion through polycarbonate filters produced single-layer vesicles with encapsulated PNPase (67% ended up inside) 5. ADP added to buffer, with or without protease 6.let react several days at RT 7.radiolabel RNA and PAGE Chakrabarti AC, Breaker RB, Joyce GF, and Deamer DW (1994). J. Mol. Evol. 39:555–559. results encapsulation leads to RNA polymerization! Pu AD ot s Pn cle AM vesi pty em sed Chakrabarti AC, Breaker RB, Joyce GF, and Deamer DW (1994). J. Mol. Evol. 39:555–559. organic material, including amphiphiles, have been found in carbonaceaous chondrites OH O monocarboxylic acids up to C10 polyaromatic hydrocarbons (PAHs): naphthalene phenanthracene anthracene Dave Deamer: liposome research phospholipids extracted from meteorites can form vesicles rehydration of organic extracts from meteorites can produce small vesicles Deamer (1997). Microb. Mol. Biol. Rev. 61:239–261. Jack Szostak: protocell research artificial cells can be made from a variety of materials methods 1.made six types of vesicles, varying the fatty acids and hence the phospholipids 2.incorporated dye into the vesicles at the same time: 5-carboxyfluorascein or calcein 3.checked for size & leakage using spectrofluorimetry and dynamic light scattering 4.put vesicles into various sugar solutions 5.conducted shrink-swell experiments using stopped-flow spectrofluorimetry 6.calculated the permeability coefficient for each sugar Sacerdote & Szostak (2005). Proc. Natl. Acad. Sci. USA 102:6004–6008. results shrink-swell experiments: Sacerdote & Szostak (2005). Proc. Natl. Acad. Sci. USA 102:6004–6008. conclusions: why is ribose superior? 1. ribose prefers furanose form (furanose more hydrophobic than pyranoses) 2. furanoses much more flexible than pyranoses 3. α-pyranose form of ribose has hydrophobic face (also compare Ps of erythrose and threose) Sacerdote & Szostak (2005). Proc. Natl. Acad. Sci. USA 102:6004–6008. Jack Szostak (Harvard): making artificial cells with life-like properties Movie compartmentalization in vitro evolution in vitro selection (test-tube evolution) Evolution Amplification Mutation Selection selection scheme phenotype assay Joyce (2007) ACIE in vitro evolution (Systematic Evolution of Ligands by Exponential Enrichment) rough numbers • what can be selected: RNA, DNA, proteins 12 – 1016 molecules original pool (G ) size: 10 0 • • mutation methods: ➡ error-prone PCR ➡ “mutator oligos” ➡ errors in non-amplifying replication ➡ environmental stress (UV, mutagens, etc.) • selection strategies ➡ binding ➡ tagging ➡ size ➡ other sequence attributes • number of generations needed to get a “winner”: about 6 creating G0 selecting winner(s) amplifying winner(s) the polymerase chain reaction (PCR)! • if you are working with DNA, PCR directly • if you are working with RNA, turn RNA into DNA first using reverse transcriptase (RT) • if you are working with proteins, PCR the gene for the protein (or make virus do it: phage display) the polymerase chain reaction (PCR) extract genomic DNA design primers do PCR reaction amplification! the polymerase chain reaction (PCR) 1967: Gobind Khorana, comes up with the idea of replicating DNA in vitro 1983: Kery Mullis, working at Cetus, develops the idea of using Taq DNA polymerase and thermal cycling 1985: Randall Saiki et al. publishes the first actual report of PCR in Science 1993: Mullis wins the Nobel Prize in Chemistry for PCR the polymerase chain reaction (PCR) but let’s go back to the 60’s bacteriophage Qβ replicase gene: codes for an RNA-dependent RNA replicase protein that copies the 3300 nt phage genome Sol Spiegelman (1967) Proc. Natl. Acad. Sci USA (1967) 58, 217–224 Sol Spiegelman (1967) in vitro (“extracellular”) serial transfer experiments Qβ RNA Qβ replicase nucleotides buffer original wild-type Qβ stock 20 minutes 20 minutes 20 minutes 20 minutes etc. assay RNA for genotype and phenotype result #1 – continuous growth of RNA etc. result #2 – infectivity drops over time etc. result #3 – some sort of sequence evolution is happening etc. result #4 – selection for much shorter RNAs! original sequence: 3300 nt etc. evolved sequence: 550 nt later experiments: resistance to ethidium bromide or RNase etc. 1980’s: along comes the PCR selection for aptamers (SELEX) selection of a ribozyme that can cleave DNA as well as RNA (selection of a ligase ribozyme) evolution of a ligase ribozyme (selection of a polymerase ribozyme) etc. etc. selection of a DNAcleaving ribozyme selection strategy Beaudry & Joyce (1992) Science 257: 635–641 selection of a DNAcleaving ribozyme mutations of wildtype = G0 the Tetrahymena group I intron (self-splices in vitro) Beaudry & Joyce (1992) Science 257: 635–641 selection of a DNAcleaving ribozyme G0 G3 G6 phenotype G9 genotype Beaudry & Joyce (1992) Science 257: 635–641 selection of the class I ligase ribozyme 14 rounds of in vitro selection b201 ligase (Bartel & Szostak, 1993) continuous evolution of the ligase ribozyme class I ligase ribozyme continuous evolution of the ligase ribozyme class I ligase ribozyme In vitro selection of the original replicase ribozyme (2001) class I ligase ribozyme Johnston et al. (2001) Science 292, 883-896. Putting it all together The Chemical Origins of Life • the molecular biologists’ dream: “imagine a pool of activated ß-D-nucleotides ...” • the prebiotic chemists’ nightmare: “monomers, polymers, chirality, information, tar ...” the big bang The Chemical Origins of Life RNA/protocells DNA the “universal” genetic code LUCA bacterial, etc., “life”