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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
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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
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”