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
CHEM 460 / 560Prebiotic Chemistry
Dr. Niles LehmanDepartment of ChemistryPortland State University
http://web.pdx.edu/~niles/Lehman_Lab_at_PSU/Chem_460_560.html
The Timeline of Life
Joyce (2002) Nature 418, 214-221
(see P&G Fig. 2.1)
timelines:
15 bya 10 bya 5 bya 0 bya13.7
BIG BANG
4.6
originof the sun
4.55
originof the Earth
ORIGINS OF LIFE ON THE EARTH
4.0 2.5
eubacteria/archaea split
0.6
origins of multicellularity
What is Life?
LIFE = “a self-sustaining chemical system capable of darwinian evolution” (Joyce/NASA)
• growth• response to stimulus• metabolism• reproduction• evolution
the “list of characteristics” approach:
life
non-life
the non-life-to-life transitionat 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
a necessary, but not sufficient, requirement for “life”
2MnO4++ + 5H2C2O4 + 6H30+ 2Mn++ + 10CO2 + 14H2O
add Mn++
the chemistry of lifethe life on the Earth is based on Carbon
Catomic number = 6electronic configuration: 1s2, 2s2, 2p2
atomic mass = 12.011isotopic 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 universefor 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 lowest
water is high
the three “stages” in the evolution of life
1. chemical evolution2. self-organization3. biological evolution
life can be considered a “negentropy machine”
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.
hν
heatΔS < 0
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
strongnuclearforce
weaknuclearforce
electro-magnetic
force
gravita-tionalforce
>> >> >>
holdsnuclearparticles together(p + n)
responsiblefor radioactive
decay(n p + e–)
holdselectronsto nuclei
(CHEMISTRY)
holdsmatter
together into largerstructures
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
p = 1Hp + p D
D + p 3He3He + 3He 4He + 2p
“Big Bang nucleosynthesis”
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 distributionsled 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
the Bethe & Weizsacker carbon cycle
Sun: T = 16 million K
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, Feouter, gaseous planets: H2, He, NH3, and CH4
formation of Earth’s moon
massive collision at 4.51 +/– 0.01 byawas another key event in the origins of life
the history of large impacts on the Earth and Moon
red: impacts on Moon blue: impacts on the Earth
moon-formationimpact
habitable zones
galactic habitable zonesolar 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”
• 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
> 120 organic molecules have been detected to date,
mostly by microwave spectroscopy(Benner, 2009)
hydrogen cyanideformaldehyde
acetaldehyde
glycoaldehyde
relative abundances of molecules in space
Small Molecule PrecursorsFound in space: Found in comets &
meteorites:• hydrogen cyanide (HCN)• acetlyene (HC CH)• formic acid (HCOOH)• formaldehyde (H2CO)• acetic acid (CH3COOH)• ammonia (NH3)• water
• amino acids• nucleobases• lipids• PAHs• water
abundant on early Earth: hydrogen sulfide, CO, water, methane, salts, etc. ...
but how?
the Earth’s early atmosphere
(contemporary atmospheres of Venus, Earth, and Mars: Zubay Table 5-2)
• 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 H2 occurred gradually but steadily
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 & N2, 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)
banded iron
4Fe(II)O + O2 2Fe2(III)O3
four key reactions could have occurred in this type of atmosphere:
1. CO2 + 2H2 H2CO + H2O abiotic formaldehyde
2. N2 + hν 2N nitrogen photolysis
3. CO2 + 2H2O CH4 + 2O2 abiotic methane
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 proto-planetary 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
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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
amino acids
fatty acids nucleotides
NH2
O
OHN
NN
N
NH2
O
OHOH
HHHH
OP-O
O-
O
sugars
O
H
HO
H
HO
H
OHOHH H
OH
OOH
the source of monomers - e.g., amino acids
a “dead” bag of chemicals
glycine, an amino acid
H2 + NH3 + CH4 + H2O H2N – CH2 – COOH energy
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 atmospherewould 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
English edition, first published in 1938
• 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
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:
1. 3Mg + N2 Mg3N2; 2Al + N2 Al2N2; 2Fe + N2 2FeN 2. FeN + 3H2O Fe(OH)3 + NH3
Δ Δ Δ
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
Geoffrey Zubay: “The synthesis of sugars in the prebiotic world is likely to have started with formaldehyde”
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
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
The Miller-Urey spark-discharge experiments
a “dead” bag of chemicals
glycine, an amino acid
H2 + NH3 + CH4 + H2O H2N – CH2 – COOH energy
the source of monomers - amino acids
the Miller-Urey spark-discharge experiments (1953-2000)
glycine, alanine, aspartic acid, etc.
Miller (1953) Science 111:528–529.
The original Miller-Urey Experiment (1952)
CH4 (20 torr) + NH3 (20 torr) + H2 (10 torr) + H2O (vapor)
2000 V spark; one-week incubation time
500 mL flask: water (“ocean”) + 2 L flask: gas (“atmosphere”)
paper chromatography
The original Miller-Urey Experiment (1952)
CH4 + NH3 + H2O + H2 + energy :
glycine > α-alanine > α-amino-n-butyric acid > β-alanine > glutamic acid > aspartic acid
= Table 4.2 in P&G
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
subsequent Miller-Urey experiments (1953–)
varied the input gasses & concentrations all the way from strongly reducing (best yields) to mildly oxidizing (poorer yields)
varied energy source (e– vs. UV vs. heat, etc.) & time
varied flask configurations and gas pressure
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
= P&G Fig. 4.4
intermediates in Miller-Urey experiments
the appearance and then disappearance of HCN and aldehydes reveals that they are key intermediates
2000V: produces free radicals to drive production of intermediates
variant Strecker synthesis of amino acids and hydroxy acids
1. the production of a cyanoamine:
RCH=O + NH3 + HC N RCHNH2C N 2. the hydration of the cyanoamine to give an amino acid:
RCHNH2C N +2H2O R–C–COOH
NH2
H
0. the production of aldehydes and HCN via free-radical chemistry from simple gaseous starting materials, for example:
a) CH4 + H2O H2CO + H2 [CH4 + e–* CH3 + H+]b) 2CH4 + N2 2HCN + 3H2
[N2 + e–* 2N ]
the classic Strecker synthesis of amino acids
the Strecker synthesis of amino acids and hydroxy acids1. 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 N (hyrdoxynitriles): used in amino acid abiosynthesis
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
5!5!
September 28, 1969Murchison, Australia
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 α-amino-n-butyric acid, along with
a range of hydroxy acids
the Miller-Urey experiments have produced at least17 of the 20 or so proteinaceaous amino acids
the three aromatics, Tyr, Trp, and Phe
require an alternative synthetic route
some require subsequent
modifications
Miller has proposed an abiotic route to histidinethat mimics the biosynthetic route
erythrose would come from the formose reaction (coming soon!)
“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 bricks out of which multifarious kinds of protein are built. 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. Miller is elated, and so is Professor Urey, his mentor.”
Miller’s experiment generated instant media attention
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 N (hyrdoxynitriles): used in amino acid abiosynthesis
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
ribose requires 5 carbons, C-O bonds, and correct stereochemistry
OHOH OH
OHO
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
The formose reaction (autocatalytic)
ribose
formaldehyde
glycoaldehyde
DL-glyceraldehyde
the formose reaction
OHOH OH
OHO
Butlerov (1860): formaldehyde + water + calcium hydroxide + heat gives a mixture of sugars
formaldehyde is used to make glycoaldehyde, trioses, and tetroses; pentoses such as ribose are made by the condensation of glycoaldehyde and a triose
the formose reactionoptimal: 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
glycoaldehyde
DL-glyceraldehyde
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
C2: glycoaldehyde
C3: DL-glyceraldehyde
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
the glycoaldehyde cycle
= Fig. 4.7 P&G
ribose is but one of many possible 5-carbon sugars:
5C
6C
4C
3C
then the straight-chain form must cyclize:
(6C example)
The formose reaction produces a dizzying array of products
Decker, Schweer, & Pohlmann (1982) J. Chromatogr. 244: 281–291.
GC
ribose
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
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)
Zubay, 1998
Steve Benner: use borate!
Borate ions can stabilize glyceraldehydes, preventing them from acting as nucleophiles and thus stemming out-of-control polymerization
glycoaldehyde + DL-glyceraldehyde pentoses as majorityCa(OH)12
boron mineral
OHO O
OHO
B
O O
Ricardo, Carrigan, Olcott, & Benner (2004) Science 303, 196
O
H
HO
H H
ulexiteNaCaB5O9•8H2O
Jack Szosak: use cell membranes!
using certain phospholipid membranes in artificial cells results in a greatly increased permeability to ribose vs. other pentoses and sugars
Sacerdote and Szostak (2005).Proc. Natl. Acad. Sci. USA,102:17–22.
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
p-RNA: pyranose RNA
TNA: threose nucleic acid
Joyce (2004)
maybe ribose came later, and simpler backbones came first:
PNA: peptide nucleic acid
GNATNA p-RNA
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
the Oró HCN polymerization experiments (1961-)
15 atoms & 50 electrons:5 C-H bonds5 C-N bonds
present in interstellar medium
15 atoms & 50 electrons:2 C-H bonds9 C-N bonds3 N-H bonds1 C-C bond
present in living systems
recombinationC NH
N
NNH
N
NH2
5 HH
hydrogen cyanide (HCN) adenine
read P&G’s discussion of HCN on the Earth
(pp. 95-97)
the mechanism of Oró HCN polymerization
HCN
adenine
1. dimerization of HCN2. trimerization to aminomaleonitrile3. tetramerization to DAMN4. UV-induced isomerization5. final HCN addition and ring closure
“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
1. dimerization of HCN2. trimerization to aminomaleonitrile3. tetramerization to DAMN4. UV-induced isomerization5. final HCN addition and ring closure
optimum rate at pH 9.2 (pKa of HCN)
= P&G Fig. 4.9
iminoacetonitrile
the mechanism of Oró HCN polymerization
1. dimerization of HCN2. trimerization to aminomaleonitrile3. tetramerization to DAMN4. UV-induced isomerization5. final HCN addition and ring closure
= P&G Fig. 4.9
the mechanism of Oró HCN polymerization
1. dimerization of HCN2. trimerization to aminomaleonitrile3. tetramerization to DAMN4. UV-induced isomerization5. final HCN addition and ring closure
Zubay: last HCN addition may come after a formylation instead, akin to purine
biosynthesis
Adenine Guanine
biosynthesis of purines
AICA equivalent
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...
Leslie Orgel: hypoxanthine + D-ribose + Mg2+ gives β-inosine under dehydrating conditions (low yield)
NH
N
N
O
N
O
OH
HH
HHOH
OP-O
O
O-
this reaction does not work for the pyrimidines!
IMP
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
There is evidence that schreibersite, when dissolved in water, can form pyrophosphate, which can phosphorylate sugars (Matt Pasek, U. Arizona)
schreibersite is a rare iron-nickel phosphide mineral, but is common in iron-nickel meteorites
phosphates from more reduced forms of P
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?!?
Powner, Gerland, and Sutherland (2009) Nature 459, 239–242
cyanamide 8+
cyanoacetylene 7+
glycoaldehyde 10+
glyceraldehyde 9+
inorganic phosphate***
arabanose amino-oxazoline 12
β-D-ribocytidine 2´,3´ phosphate
(oh yeah!)
“the prebiotic synthesis of activated pyrimidinenucleotides should be viewed as predisposed”
Powner et al. (2009) Nature 459, 239–242
“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.”
Powner, Gerland, and Sutherland (2009) Nature 459, 239–242
a three-fer!
1M phosphate buffer, pH 7, 40˚C, o/n
movie
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
• polymerizing monomers with the liberation of water ... in water!
Condensation
H2N CH C
CH3
OH
O
H2N CH C
CH2
OH
O
OH
HN CH C
CH2
OH
O
OH
H2N CH C
CH3
O
+
+ H2OAla Ser
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)
Nature 129: 1221–1223 (1959)
Sydney Fox’s proteinoids (debunked)
Science 201: 67–69 (1978)Thermal condensation on clay
Lahav, N., White, D., Chang, S.
J.Mol. Evol. 17: 285–294 (1981)
Cyanamide-mediated polymerization
(draw mechanism on whiteboard)
The RNA World...
...needs ribose, nucleobases, and phosphates ... and chains!
RNA structure
Azoarcus ribozyme (205 nt)Adams et al. (2004) Nature 430, 45-50.
5´-GUGCCUUGCGCCGGGAAACCAC...-3´
The Catalytic Repertoire of RNA
Chen, Li, & Ellington (2007)
The Source of Polymers
N
NN
N
NH2
O
OHOH
HHHH
OP-O
O-
O
'5 3'A A A
• activation is needed: triphosphate, imidizole, etc.• linakage geometry is important• templating can help
contemporary polymerases
Figure 30-10 Schematic diagram for the nucleotidyltransferase mechanism of DNA polymerases.
in-line nucleophilic attack
abiotic RNA polymerization
1. high-energy condensing agents1.1. amino acid adenylates1.2. imidizolides1.3. water-soluble carbodiimides1.4. purines and pyrimidines
2. catalytic action2.1. inorganic ions2.2. clays2.3. oligonucleotide templates2.4. ribozymes2.5. lipids
amino acid adenylates
nucleotides have been proposed to condense amino acids,so can the reverse be true: AA used to condense nt’s?
O
N
NN
N
NH2
O
OHOH
HHHH
OPO
O-
ONH2
imidazolides
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 aphosphorus center
N
NN
N
NH2
O
OHOH
HHHH
OP
O-
O
N N
R
R = H or CH3
–HO:
ImpA
see P&G, Fig. 4.16
water-soluble carbodiimides
phosphoramidite
R1–N=C=N–R2
example: EDC = 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide
purines and pyrimidines
purine- and pyrimidine-like molecules are attachedto the 5´ phosphate and serve as good leaving groups
N
NN
N
NH2
O
OHOH
HHHH
OP
O-
O
NNN
NN
N
NH2
O
OHOH
HHHH
OP
O-
O
N
N
NN
H2N
H3C
4-dimethylaminopyridinium-AMP
adenosine-5´-phophoro-1-methyladeninium
catalysts for RNA condensation:points to consider
1. template-directed vs. non-template directed2. 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 self-condensation 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
Self-complementary autocatalysis has been previously demonstrated, but nucleic acid replication utilizes complementary strands,
which can replicate via cross-catalysis
A = CCGB = CGG
example study #2:Sievers & von Kiedrowski (1994) Nature 369: 221–224
example study #2:Sievers & von Kiedrowski (1994) Nature 369: 221–224
the addition of a particular product enhanced the rate of synthesis of that one product only
AB
BA, AA, and BB
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!
example study #3:Ferris et al. (1996) Nature 381: 59–61
Ferris et al. (1996) Nature 381: 59–61
shorter RNA chains
longer RNA chains
Jim Ferris: daily “feeding” of nucleotides to clay results in RNA chains!
the correct linkage and stereochemistry can be achieved
Joshi, Aldersley, Zagorevskii, & Ferris (2012) Nucleosides, Nucleotides, & Nucleic Acids, in press
Clays: layers of ionsexample: 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
• how do you transfer information from one molecule to another?
• balance between fidelity (for information maintenance) and errors (for evolution)
RNA making RNA:self-replication
+ + – –
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
ribozymesphosphoester bond cleavage
(hydrolysis)
2´ -OH attack
trans-esterification
trans-esterification
3´ -OH attack
self-cleaving ribozymes & reversibilitythis
molecule should look
familiar!
group I intronribozyme
Azoarcus ribozyme (205 nt)Adams et al. (2004) Nature 430, 45-50.
in vitro selection (test-tube evolution)
Joyce (2007) ACIE
selection scheme
phenotypeassay
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
b201 ligase (Bartel & Szostak, 1993)
NNNN–OH + pppN
:
Johnston et al. (2001) Science 292, 883-896.
In vitro selection of the original replicase ribozyme (2001)
class I ligase ribozyme
replicase-14
primer (orange) + template (red)
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
Zaher & Unrau (2007) RNA 13, 1017-1026.
In vitro selection of an improved replicase ribozyme (2007)
in vitro selection
replicase-14
water-in-oil emulsions
Zaher & Unrau (2007) RNA 13, 1017-1026.
In vitro selection of an improved replicase ribozyme (2007)
replicase-20
up to 20 nt, with 3–4-fold more accuracy
Wochner et al. (2011) Science 332, 209-212.
In vitro selection of an even more improved replicase ribozyme (2011)
the tC19Z ribozyme (replicase-95) can
polymerize up to 95 nt!95/187 = 50%
up to 95 nt, but only certain templates
replicase-95
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 self-replicating 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 = 0.033νmax = 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
abiotic material is achiral or racemic
Text
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 enantiomerschiral 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
7. The compartmentalization problem
metabolism FIRST?
Metabolism-first Theoriesthe 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 worldDe Duve has proposed thioesters as a key molecule
to allow the build-up of larger molecules
De Duve: “without additional help of both catalytic and energetic nature, the prebiotic broth would have remained
sterile”
R1 S C R2O
origin of thioesters
e.g., H2S would have been abundant on the prebiotic Earth,and simple carboxylic acids could have derived from Miller-
Urey type reactions
R1 S C R2O
R1 SH R2 C OH
O
thiolcarboxylic
acidthioester
+energy
H+
origin of thioesters in a hot acidic environment
the thiol group in thioesters is quite transferable
thioester-dependent reductions
R1 S C R2O
thiolreducing power
thioester
+
aldehyde
+2H+ + 2e– R1 SH R2 C HO
the thiol group in thioesters is quite transferable
thioester-dependent phosphorylations
R1 S C R2O
thiolinorganic phosphatethioester
+
acyl phosphate
+R1 SHHO P OO
OHO P O
O
OHCO
R2
the thiol group in thioesters is quite transferable
thioester-dependent catalytic production of multimers
thiolthioestercarriers
+
dimer
+
R' S C R1
O
R' S C R2
O R' S C R2
O
R1 R' SH
De Duve: thioesters were used for general activation and sequential group transfer
from “Blueprint for a Cell” (1991)
the pyrite world
Wächtershäuser views metabolism as primitive, and “inventing” a genetic structure later to maintain itself
hydrogen sulfide, in combination with the two redox states of iron, could have provided the
functional precursors of all extant biochemicals
FeS + H2S 2H+ + 2e– + FeS2reducing power
pyriteiron
sulfidehydrogen
sulfide
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
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
FeS + H2S H2 + FeS2
CO2 + H2 HCOOH
FeS + CO2 + H2S HCOOH + FeS2
carbon monoxide can be converted to acetic acid
then the carbonylated Fe-S intermediate can be “desulfurized” to generate acetic acid and pyruvate:
2FeS + 6CO + 2R-SH 2S0 + H2 + Fe2(RS)2(CO)6
first, iron sulfide is carbonylated:
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
FeS/H2S might be able to reduce the relatively oxidized (electron-poor) hydrocarbons such as acetylene that are
present in the interstellar dust
(draw scheme on whiteboard)
the TCA cycle: at the root of
anabolism
the cycle traces both the number of carbons
and their relative oxidation states
“all extant organisms oxidize chemical fuels” to generate reducing power for metabolism
generates reducing power
the reductive TCA cycle:
carbon fixation ... performed by protein enzymes containing
Fe-S clusters!
reducing powerused to fix
inorganic carbon
the acetyl CoA pathway portion= the direct formation of acetate from CO2 or CO
in biology, this is catalyzed by the
acetyl-CoA synthase enzyme complex ... using an Fe-S cluser
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 acetyl-CoA 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 evolution2. self-organization3. 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
Nature 129: 1221–1223 (1959)
Sydney Fox’s proteinoids (debunked)
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
liposomesmonolayers 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 acidslong aliphatic
hydrocarbon chains, with or without
unsaturated C–C bonds
amphipathic molecules “self-assemble”
lipid synthesis – today1. make fatty acid side chains 2. esterify side chains to polyol
lipid synthesis – abiotic
Fischer/Tropsch reaction
1. make side chains2. esterify side chains to polyol
C + H2O CnH2n+2 Fe, Ni
Δ
addition of successive CO units
lipid synthesis – abiotic1. make side chains2. esterify side chains Wachtershauser’s proposal
CH2O CH2 = CH2 FeS2 / H2S
Δ(100˚C, pH7)
lipid synthesis – abiotic1. make side chains2. 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
...dehydration & rehydration
1. make side chains2. esterify side chains to polyol
glycerol + FA + phosphate, then ...
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
Chakrabarti AC, Breaker RB, Joyce GF, and Deamer DW (1994). J. Mol. Evol. 39:555–559.
1.the lipid DMPC (dimyrisoyl phosphatidyl choline) was sonicated in water
2.dry PNPase added & mixture dried under N2 gas
3.rehydration in buffer4.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 RT7.radiolabel RNA and PAGE
results
Chakrabarti AC, Breaker RB, Joyce GF, and Deamer DW (1994). J. Mol. Evol. 39:555–559.
encapsulation leads to RNA polymerization!
AM
P not AD
P used
empty vesicles
organic material, including amphiphiles, have been found in carbonaceaous chondrites
naphthalene
polyaromatic hydrocarbons (PAHs):
phenanthracene
anthracene
monocarboxylic acids up to C10
O
OH
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
Sacerdote & Szostak (2005). Proc. Natl. Acad. Sci. USA 102:6004–6008.
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 solutions5.conducted shrink-swell experiments using
stopped-flow spectrofluorimetry6.calculated the permeability coefficient for each
sugar
results
shrink-swell experiments:
Sacerdote & Szostak (2005). Proc. Natl. Acad. Sci. USA 102:6004–6008.
conclusions:why is ribose superior?
Sacerdote & Szostak (2005). Proc. Natl. Acad. Sci. USA 102:6004–6008.
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)
compartmentalization
Movie
Jack Szostak (Harvard):making artificial cells with
life-like properties
in vitro evolution
in vitro selection (test-tube evolution)
Joyce (2007) ACIE
selection scheme
phenotypeassay
EvolutionEvolution
Amplification
Mutation
Selection
in vitro evolution
(Systematic Evolution of Ligands by Exponential Enrichment)
rough numbers• what can be selected: RNA, DNA, proteins• original pool (G0) size: 1012 – 1016 molecules• 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 primersdo PCR reaction
amplification!
the polymerase chain reaction (PCR)
1983: Kery Mullis, working at Cetus, develops the idea of using Taq DNA
polymerase and thermal cycling
1993: Mullis wins the Nobel Prize in Chemistry for PCR
1967: Gobind Khorana, comes up with the idea of
replicating DNA in vitro
1985: Randall Saiki et al. publishes the
first actual report of PCR in Science
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 RNAQβ replicasenucleotides
buffer
20 minutes 20 minutes 20 minutes 20 minutes
etc.
assay RNA for genotype and phenotypeoriginal wild-type
Qβ stock
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!
etc.
original sequence:3300 nt
evolved sequence:550 nt
etc.
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.
selection of a DNA-cleaving
ribozyme
Beaudry & Joyce (1992) Science 257: 635–641
selection strategy
selection of a DNA-cleaving
ribozyme
Beaudry & Joyce (1992) Science 257: 635–641
the Tetrahymena group I intron (self-splices in vitro)
mutations of wildtype = G0
selection of a DNA-cleaving
ribozyme
Beaudry & Joyce (1992) Science 257: 635–641
G0 G3
G6 G9
phenotype genotype
selection of theclass I ligase ribozyme
b201 ligase (Bartel & Szostak, 1993)
14 rounds of in vitro selection
continuous evolution of the ligase ribozyme
class I ligase ribozyme
continuous evolution of the ligase ribozyme
class I ligase ribozyme
Johnston et al. (2001) Science 292, 883-896.
In vitro selection of the original replicase ribozyme (2001)
class I ligase ribozyme
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 Chemical Origins of Life
DNA
LUCA
bacterial, etc., “life”
the “universal” genetic code
RNA/protocells
the big bang