CHEM 460 / 560 Prebiotic Chemistry

transcript

CHEM 460 / 560Prebiotic Chemistry

Dr. Niles LehmanDepartment of ChemistryPortland State University

niles@pdx.edu

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

originof the sun

originof the Earth

ORIGINS OF LIFE ON THE EARTH

4.0 2.5

eubacteria/archaea split

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:

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.

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 Big Bang

13.7 bya

the four fundamental forces in Nature

strongnuclearforce

weaknuclearforce

electro-magnetic

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

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”

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 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”

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

Kelly Scientific Resources has an opening for an Extractions Technician at an environmental company in Tigard, OR. This position has the potential to become a temp to hire opportunity. The successful candidate will perform sample preparation duties including solvent extractions, solid phase extractions and clean-up techniques using EPA methods. Schedule is a late day shift starting around 10am till about 6pm.

Kelly Scientific Resources has an opening for a Sample Receiving Technician at an Environmental Company in Tigard, OR. This position has the potential to become a temp to hire opportunity. Individual will be responsible for sample collection, shipping, receiving, technical data entry, and record keeping of samples received. Will also provide customer service to clients on the phone and in person.

Kelly Scientific Resources has an opening for a Quality Assurance Technician at a Chemical Manufacturing company in Tacoma, Washington. The successful candidate will be responsible for analytical and physical testing of products produced within the production facility, monitoring of critical quality systems, reporting and documentation, as well as other general laboratory duties.

Senior Lab Technician - Kelly Scientific Resources has an opening for a Chemical Lab Technician at a chemical manufacturing company in Hillsboro, Oregon on a temp-hire basis.

entry-level jobs

If interested, please contact Kelly Scientific Resources directly at 503-245-4533 or send your resume to 5025@kellyservices.com. Kelly Scientific is a division of Kelly Services - an international, Fortune 500 Company with a reputation for excellence. Kelly Scientific is devoted to partnering with companies to provide scientific professionals with the best possible positions to meet their career goals. There is never a fee to our applicants or employees. For more information, visit www.kellyscientific.com.

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

sugars

OHOHH H

the source of monomers - e.g., amino acids

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)

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 source of monomers - amino acids

The Miller-Urey spark-discharge experiments

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

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

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

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

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

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

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

glycoaldehyde

the glycoaldehyde cycle

= Fig. 4.7 P&G

ribose is but one of many possible 5-carbon sugars:

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.

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

Ricardo, Carrigan, Olcott, & Benner (2004) Science 303, 196

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 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

hydrogen cyanide (HCN) adenine

read P&G’s discussion of HCN on the Earth

(pp. 95-97)

the mechanism of Oró HCN polymerization

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)

optimum rate at pH 9.2 (pKa of HCN)

= P&G Fig. 4.9

iminoacetonitrile

= P&G Fig. 4.9

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)

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

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

• polymerizing monomers with the liberation of water ... in water!

Condensation

H2N CH C

HN CH C

H2N CH C

+ 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

'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?

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

R = H or CH3

–HO:

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

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

templates: pre-existing polymer templates have been demonstrated empirically to speed up RNA

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:

Self-complementary autocatalysis has been previously demonstrated, but nucleic acid replication utilizes complementary strands,

which can replicate via cross-catalysis

A = CCGB = CGG

the addition of a particular product enhanced the rate of synthesis of that one product only

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.”

• 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

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)

template extension by replicase-14

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

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)

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

thiolcarboxylic

acidthioester

+energy

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

thioester-dependent phosphorylations

R1 S C R2O

thiolinorganic phosphatethioester

acyl phosphate

+R1 SHHO P OO

OHO P O

thioester-dependent catalytic production of multimers

thiolthioestercarriers

R' S C R1

R' S C R2

O R' S C R2

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 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

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

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.

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!

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

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

results

shrink-swell experiments:

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)

compartmentalization

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!

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

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

assay RNA for genotype and phenotypeoriginal wild-type

Qβ stock

result #1 –continuous

growth of RNA

result #2 –infectivity drops

over time

result #3 –some sort of

sequence evolution is happening

result #4 –selection for much

shorter RNAs!

original sequence:3300 nt

evolved sequence:550 nt

later experiments:resistance to

ethidium bromide or RNase

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

ribozyme

the Tetrahymena group I intron (self-splices in vitro)

mutations of wildtype = G0

ribozyme

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

In vitro selection of the original replicase ribozyme (2001)

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

bacterial, etc., “life”

the “universal” genetic code

RNA/protocells

the big bang

CHEM 460 / 560 Prebiotic Chemistry

Documents