IRENA MAMAJANOV FROM MESSY CHEMISTRY TO THE ORIGINS OF LIFE Habitability: Producing Conditions Conducive to Life LPI “First Billion Years” Conference Series September 9 2019 PLANETARY HABITABILITY AS PERCEIVED BY A CHEMIST WHAT IS HABITABILITY ANYWAY? Planetary habitability is the measure of a planet's or a natural satellite's potential to develop and sustain life. EARTH-CENTRIC? DEFINITION OF LIFE

LIFE IS A SELF-SUSTAINING SYSTEM CAPABLE OF DARWINIAN EVOLUTION

NASA Working Definition HOW WE STUDY ORIGINS OF LIFE

TWO APPROACHES IN THE BROADEST SENSE

▸ More Earth biology-centric

▸ Prebiotic synthesis of biological building blocks

▸ Setting biological processes in abiotic environments

▸ Evolution of biological structures

▸ More open-ended: Building a chemical system capable of Darwinian Evolution

▸ Selectivity

▸ Replication

▸ Heredity MORE EARTH BIOLOGY-CENTRIC: UNSATISFYING? TV PARADOX ? ? “MORE OPEN ENDED” APPROACH ▸ Looking at systems level processes. MESSY CHEMISTRY ▸ “Systems Chemistry” usually = small defined networks

▸ “Messy Chemistry” = the network chemistry of large, “intractable”, prebiotically plausible systems.

▸ Sloppy biological processes

▸ Processes resembling biological but inefficient

Small fraction of the Organic Chemistry M. Kowalik, C.M. Gothard, A.M. Drews, N.A. Gothard, B.A. Grzybowski, K.J.M. Bishop, Parallel optimization of synthetic pathways within the network of organic chemistry. Angew. Network (~0.001%). Chem. Int. Ed. 51, 7928-7932 (2012). EVOLUTION OF THE CHEMOSPHERE AND BIOCHEMICAL NETWORKS

Biomimetic Systems

Systems approximating biological function • Protoenzymes • Protocells

Open-Ended Systems

Systems having no predetermined limit or boundary • Autocatalytic systems

M. Kowalik, C.M. Gothard, A.M. Drews, N.A. Gothard, B.A. Grzybowski, K.J.M. Bishop (2012) Angew. Chem. Int. Ed. 51:7928-7932 Small fraction of the Organic Chemistry Network (~0.001%). Metabolic Pathways: http://pathview.r-forge.r-project.org/ MODEL SYSTEM

POLYESTERS IN PREBIOTIC CONTEXT FUNCTIONAL POLYMERS POLYESTER LIBRARY

Lac Iso Phe Met Glc 20 + + + + 5 Wet/ dry cycle Unique sequences

43500 unique Glc2-Lac0-Phe0-Iso1-Met5 sequences identified Met3 - Glc3-Lac9-Phe2-Iso0-Met5 Iso0 - 6 Glc3-Lac1-Phe0-Iso1-Met2 Phe2 - Lac1 - 0

Glc Glc0-Lac6-Phe0-Iso0-Met7 Intensity x 10 Glc1-Lac7-Phe2-Iso2-Met7

Glc1-Lac1-Phe4-Iso0-Met10

Chandru K, Guttenberg N, Giri C, Hongo Y, Butch C, Mamajanov I, Cleaves HJ. (2018). Simple prebiotic synthesis of high diversity dynamic combinatorial pH 3, 80 C, POS mode (FTICR-MS) M/Z polyester libraries. Communications Chemistry 1:30 FUNCTIONAL POLYMERS

ORGANIZATION THROUGH COMPARTMENTALIZATION

▸ Fatty acid vesicles (Szostak lab, ▸ Coacervates (Oparin, Haldane) Deamer lab)

▸ Membraneless polymer microdroplets FUNCTIONAL POLYMERS POLYESTER MICRODOPLETS

Tony Jia

Kuhan Chandru

Scale bars 100 µm, insets 10 µm Jia, Chandru, et al. in press FUNCTIONAL POLYMERS POLYESTER MICRODOPLETS sfGFP

Scale bar 100 µm Jia, Chandru, et al. in press FUNCTIONAL POLYMERS CAN MESSY POLYMERS BE FUNCTIONAL? • The Good

• Few papers describe the catalytic activity Catalytic Microspheres of the microspheres, mostly towards hydrolysis reactions

• The Bad

• The catalytic activity demonstrated was only marginal

• No mechanistic explanation was provided

• The Ugly

• Unsubstantiated claims of Glutamic Acid • Non-random incorporation of amino acids

• Linearity Fox SW & Harada K (1958), Science 128:1214 Fox SW & Harada (1960), JACS 82: 3745- 51 • Life-like behavior and consciousness Fox SW (1989), J.Mol.Struct. 199: 183-8 FUNCTIONAL POLYMERS

Protoenzymatic Functions of Messy Polymers: Few Old Ideas

Prebiotic melanin Eumelanin

Part of the structural formula of eumelanin. The arrow denotes where the polymer continues

Blois M (1965), In: The Origin of Prebiological Systems and their Molecular Matrices (ed. Fox SW) PREBIOTICALLY PLAUSIBLE POLYMERS

PLAUSIBLE POLYMERIC ARCHITECTURES IN PREBIOTIC “TARS”

Mamajanov I and Herzfeld J. (2009) J Chem Phys 130, 134503

http://www.looking-glass-blog.com/2016/07/callister-chapter-14-polymer-structure.html HYPERBRANCHED POLYMERS

PROPERTIES AND APPLICATIONS OF HYPERBRANCHED POLYMERS

▸ Properties

▸ Abundance of Functional Groups

▸ Intramolecular Cavities

▸ Low Viscosity

▸ High Solubility

▸ Applications (only to mention a few)

▸ Additives (e.g. in polymer coatings)

▸ Supramolecular encapsulation agents

▸ Nanoparticle supports

▸ Drug/gene delivery agents FUNCTIONAL MESSY POLYMERS: PROTOENZYMES

Biology: Synthetic Prebiotic Enzymes chemistry: chemistry: Dendrizymes Hyperbranched Polymers MICROENVIRONMENTS “PROTOENZYME ASSAY”: KEMP ELIMINATION

Kemp elimination is a base catalyzed reaction sensitive to T = 30˚C; base: tetramethylguanadine solvent polarity. An assay based on Kemp elimination will therefore probe the microenvironment provided by the proto- enzymes. Kemp DS, Casey ML (1973), JACS 95: 6670-6674 BUILDING A PROTO-ENZYME

Methylsuccinic Acid Adipic Acid

Mamajanov I and Cody GD. (2017) Phil. Trans. R. Soc. A 2017 375 2016035 ASSAYING A PROTO-ENZYME

Me-Succinic Acid/Glycerol/TEA Polymer

Adipic Acid/Glycerol/TEA Polymer

Citric Acid/Glycerol/TEA Polymer

Monomeric TEA

Mamajanov I and Cody GD. (2017) Phil. Trans. R. Soc. A 2017 375 2016035 METAL SULFIDES IN PREBIOTIC CHEMISTRY

THE IMPORTANCE OF CONSIDERING METAL SULFIDE PROTOENZYMES

▸ Iron-Sulfur Clusters in Modern enzymes

▸ Ferredoxins, hydrogenases, nitrogenases.

▸ In Prebiotic Chemistry

▸ Iron-Sulfur World Hypothesis (G. Wachtershauser)

▸ Zinc World Hypothesis

▸ The hypothesis suggests that life emerged within compartmentalized, photosynthesizing ZnS formations of hydrothermal origin (the Zn world), assembled in sub-aerial settings on the surface of the primeval Earth. (A. Mulkidjanian)

▸ rTCA cycle driven by photocatalytic ZnS minerals (S. Martin) ENCAPSULATION OF NANOPARTICLES BY MESSY POLYMERS

SYNTHESIS OF NANOPARTICLES SUPPORTED BY A HYPERBRANCHED POLYMER

Polyethyleneimine (PEI)

Procedure:

1)Prepare an aqueous solution of the polymer

2)Add Zn/Co Cl2 solution, stir

3)Add Na2S solution, stir Tony Jia Rehana Afrin METAL SULFIDE NANOPARTICLE ENCAPSULATION BY MESSY POLYMERS

COS AND ZNS FORM STABLE CLEAR WATER SOLUTIONS IN THE PRESENCE OF PEI

CoS CoS/PEI CoCl2/PEI CoCl2

ZnS/PEI

ZnS

Particle size (polymer/cluster nanocomposite) < 100nm by DLS METAL SULFIDE NANOPARTICLE ENCAPSULATION BY MESSY POLYMERS

TEM ANALYSIS OF ZNS PARTICLE/HYPERBRANCHED POLYMER COMPOSITES

200nm 200nm

Fresh Sample 2 week old sample MESSY PROTOENZYME “PROTOENZYME ASSAY”: EOSIN B PHOTODEGRADATION

Irradiation w/o catalyst

Eosin B

Photo degradation monitoring TiO2 21nm NPs

0min

ZnS/G2C 15min Normalized Eosin B Concentration Normalized ZnS/PEI 30min

high-pressure Hg lamp (125 W) 45min -5 Eosin B 5.0x10 5M Total volume 30mL 120min ZnS - 10mg/ PEI (Mw= 800Da) - 280mg NETWORK PROPERTIES

CONSTRUCTING AN EVOLVABLE CHEMICAL SYSTEMS EVOLUTION

SELECTION REPLICATION HEREDITY CERTAIN TRAITS, PROPERTIES AUTOCATALYSIS HERITABLE VARIATIONS HYPOTHESIS FOR AN EVOLVABLE CHEMICAL SYSTEM

RNA WORLD

NUCLEOTIDES SELECTIVELY BIND TO TEMPLATE

RNA GETS REPLICATED

MISMATCHES CAN HAPPEN RNA WORLD?

RNA WORLD: PROBLEMS TO BE ADDRESSED (1)

▸ Formation of nucleotides

M.W.I. Nam,Powner, H.G.,B. Nam, Gerland, R.N. J.D. Zare Sutherland,, PNAS Jan Nature 2018, 115459: (1) 239–242 36-40 RNA WORLD?

RNA WORLD: PROBLEMS TO BE ADDRESSED (2)

▸ Formation of RNA

▸ Nucleotide coupling

▸ Strand separation

Aldersley, M.F.; Joshi, P.C.; Price, J.D.; Ferris, J.P. The role of montmorillonite in its catalysis of RNA synthesis. Appl. Clay Sci. 2011, 54, 1–14. NETWORK PROPERTIES

CONSTRUCTING AN EVOLVABLE CHEMICAL SYSTEMS EVOLUTION

SELECTION REPLICATION HEREDITY CERTAIN TRAITS, PROPERTIES AUTOCATALYSIS HERITABLE VARIATIONS SELECTIVE FORMOSE REACTION

BY A. RICARDO, M. A. CARRIGAN, A. N. OLCOTT, S. A. BENNER (2004) SCIENCE 303 : 196 “Organic molecules given energy and left to themselves devolve into complex mixtures, “asphalts” better suited for paving roads than supporting Darwinian evolution.”

–Steve Benner TAR PROBLEM

IS IT POSSIBLY TO SELECTIVELY SYNTHESIZE FUNCTIONAL POLYMERS AND AVOID TAR FORMATION?

HCN polymer (polymerization initiated with Rose bengal)

Black polymer formed Polymer formed in Miller-Urey System upon thermal decomposition of diaminomaleonitrile Titan as seen by (DAMN) Cassini-Huygens. Brown color attributed to polymeric material (tholin) SELECTION IN HYPERBRANCHED POLYMERS

Increasing

Degree of

Multifunctional Polymerization Polymerization (DP) monomers Hyperbranched Polymers

One-pot polycondensation reaction Flory (1952) • Not perfectly branched Hyperbranched Polymers • Low control over mass and size Gelation (solidification, • Broad molar mass distribution A2 + B3 - Approach One-pot polycondensation reaction • Irregular shape (globular, amorphousFlory (1952) decreased solubility) • Not perfectly branched structure, low viscosity) Used when AB -Monomer is difficult to synthesise (e.g. reactivity too high) 2 • Low control over mass and size • No gelation (high solubility) • Broad molar mass distribution Types of units present: • Irregular shape (globular, amorphous • Dendritic unit (both B reacted) structure, low viscosity) Danger of • Linear unit (one B reacted) • No gelation (high solubility) crosslinking Monomers• Terminating are often unit (only A reacted) Types(sol of and units gel present: commercially• Focal unit available! (A did not react, but • Dendriticformation) unit (both B reacted) both B units) present only once • Linear unit (one B reacted) •VeryTerminating little control unit (only A reacted) 2,2-Bis(hydroxymethyl)propionic acid • overFocal molar unit mass(A did not react, but andboth topology! B units) present only once

No focal unit is present HYPERBRANCHED POLYESTER SYSTEM

O OH O O OH

HO OH HO OH OH 1 : glycerol 2 citric acid B2 A3

O OH OH OH OH O O OH O O

O O HO O HO O OH O O

HO O O O HO O OH

OH TAR PROBLEM

CAN GELATION BE PREVENTED IN HYPERBRANCHED POLYESTERS BY SUBJECTING THEM TO THE WET-DRY CYCLE?

▸ Continuously dried sampled gelated after 48 hours of drying

▸ Cycled samples remained soluble after at least 8 cycles (32 days)

▸ NMR, SEC and MS analyses consistent with branched structure in the cycled sample and cross-linked in the continuously dried one

Irena Mamajanov (2019) Life, 9(3): 56 NETWORK PROPERTIES

CONSTRUCTING AN EVOLVABLE CHEMICAL SYSTEMS EVOLUTION

SELECTION REPLICATION HEREDITY CERTAIN TRAITS, PROPERTIES AUTOCATALYSIS HERITABLE VARIATIONS AUTOCATALYTIC SETS

AUTOCATALYSIS IN MESSY NETWORKS

▸ A single chemical reaction is said to be autocatalytic if one of the reaction products is also a catalyst for the same or a coupled reaction.

▸ An autocatalytic set is a collection of entities, each of which can be created catalytically by other entities within the set, such that as a whole, the set is able to catalyze its own production. In this way the set as a whole is said to be autocatalytic.

Nathaniel Virgo Jim Cleaves 2A )* A 1 2 SELF-ORGANIZING AUTOCATALYSIS IN ARTIFICIAL CHEMISTRY A +A )* A 1 2 3 A +A )* A 1 3 4 . Nathaniel . Virgo 2A )* A 2 4 A +A )* A 2 3 5

A A A A

A8 A9 A10 A7 A11 A4 A5 A7 A8 A10 A11 A13 A14 A6 A12 A1 +A1 A2 2A1 )*!A2 A5 A2 +A1 A3 A1 +A2 )*!A3 A4 2A2 A4 A1 +A3 )*!A4 A3 +A. 1 A4 final concentration . ! A3 +A2 A5 A8 A9 A10! A11 oligomer length A7 2A )* A A4 +A2 1 4 A5 concentration A12 A6 ! A2 +A3 )* A5 A5 . .

concentration A4

timetime Virgo N. et al. (2016), Artificial Life 22(2):138-152 THE SEARCH FOR AUTOCATALYTIC SETS

What we know so far:

1) Samples with the metal Simple Nitrogen Sources salt cocktail and not individual salts exhibit this behavior.

2) The color change is associated with No Cations aqueous HCN chemistry

3) Inoculating fresh solutions with a small fraction from the Transition Metal “autocatalytic” sample results in much faster Simple Carbon Sources Simple Carbon Cation Cocktail color change.

4) Work in progress: analysis is extremely challenging NETWORK PROPERTIES

CONSTRUCTING AN EVOLVABLE CHEMICAL SYSTEMS EVOLUTION

SELECTION REPLICATION HEREDITY CERTAIN TRAITS, PROPERTIES AUTOCATALYSIS HERITABLE VARIATIONS HEREDITY - AN IDEA

MOLECULAR IMPRINTING HEREDITY IN MESSY CHEMISTRY

Molecular Imprinting: The missing piece in the puzzle of abiogenesis? K. Eric Drexler Future of Humanity Institute, Oxford University [email protected] July 20, 2018 Abstract In a neglected 2005 paper, Nobel Laureate Paul Lauterbur proposed that molecular imprinting in amorphous materials—a phenomenon with an extensive experimental literature—played a key role in abiogenesis. The present paper builds on Lauterbur’s idea to propose imprint-mediated templating (IMT), a mechanism for prebiotic peptide replication that could potentially avoid a range of difficulties arising in classic gene- first and metabolism-first models of abiogenesis. Unlike models that propose prebiotic RNA synthesis, activation, and polymerization based on unknown chemistries, peptide/IMT models are compatible with demonstrably realistic prebiotic chemistries: synthesis of dilute mixtures of racemic amino acids from atmospheric gases, and polymerization of unactivated amino acids on hot, intermittently-wetted surfaces. Starting from a peptide/IMT-based genetics, plausible processes could support the elaboration of genetic and metabolic complexity in an early-Earth environment, both explaining the emergence of homochirality and providing a potential bridge to nucleic acid metabolism. Peptide/IMT models suggest directions for both theoretical and experimental inquiry. AN IDEA

IMPRINT MEDIATED PEPTIDE REPLICATION

Amino acid/small peptide

Wetting Drying

Peptide template

Drexler, K.E. Molecular Imprinting: The Missing Piece in the Puzzle of Abiogenesis? arXiv, 2018; 1807.07065v1. EVOLUTION IN CHEMICAL SYSTEMS

FEW CONCLUDING THOUGHTS

▸ Stepping away from tracing biology might give us universal life principles

▸ Messy chemical systems are where interesting, possibly emergent processes happen

▸ Bottleneck: Analytical chemistry is challenging *Photo credit Nerissa Escanlar ACKNOWLEDGEMENTS

Tony Jia* Nathaniel Virgo* Jim Cleaves*

Rehana Afrin* Melina Caudan Nicholas Guttenberg* Masahiko Hara

Kuhan Chandru* Riquin Yi* Ajay Verma