Molecular Fossils : Biomarkers

1. Molecular Fossils : Biomarkers Term ‘Biomarker’ also used in relation to health of ecosystems and humans Prognostic and Predictive Indicators

2. Readings • Hinrichs et al., Nature 1999 • Thiel et al., GCA 1999 – Both papers on linking biomarkers to AOM – Potential examination material • Fogel and Cifuentes 1994, Isotopic (esp.13C) fractionation during primary production – Potential examination material • Hayes 2002, 13C and 2H-fractionation during biosynthesis – For the really committed!!!!!!; mostly not examinable

3. Dimensions of Organic GeochemistryBased on a chemical perspective Size scales range from atomic to global, timescales to 100’s of millions yrs Generally concerns nonliving organic matter Can involve any natural environment, modern and ancient Overlaps with societal issues: petroleum exploration, fuels research, pollution, water quality, waste disposal, soil fertility, forestry, forensic chemistry, archaeology, natural products studies, drugs Why is nonliving natural organic matter important? food, fuel, structural materials, drugs plays an important role in global cycles of C, O, N, P, S, metals etc affects soil fertility and structure, redox chemistry, photochemistry, light transmission record of environmental conditions, history and reactions (molecular fossils)

4. Data richness • large number of information units = isotopes, atoms, molecules, stereochemistry • a. 1 drink of water = 250 ml, at 4 mg/L of organic matter ª 1 mg organic matter, at an average of 1000Da / molecule ª 10-6 moles of molecules, at 6 x 1023 molecules/mole ª 6x1017 organic molecules in the cup. • b. every 1000 Da molecule contains about 50% carbon ª 500 Da of carbon,at 12 Da per C atom ª 40 carbon atoms ª lots of potential for structural diversity (e.g. at least 10 structural variants for an acyclic 7-carbon hydrocarbon). • c. since ≒1/100 all carbons is 13C, about 40% of all molecules will have one 13C,that one 13C could be in up to 40 different sites the individual molecules. • d. about 1/1012 carbons in the contemporary biosphere is a 14C, although the odds of any one molecule having a 14C is less than 1/1011, there will be about 40 x 1017/1012≒ 4x105 radioactive molecules in the cup of water, decaying at a predictable rate. • e. organic matter has also: 1H, 2H, 3H, 14N, 15N, 16O, 17O, 18O, P, and 32S, 33S 34S and 36S, each of which adds to structural diversity and information richness. • every asymmetric carbon has two possible stereoisometric forms, making the maximal number of stereoisometric forms equal to 2n, where n=number of asymmetric carbons. If only 10% of the carbons in this hypothetical molecule are chiral, that molecule will have up to 16 different forms.

5. Organic matter traces biological and geographic sources “biomarkers”: 1. vanillin  lignin  vascular plants  terrigenous origin 2. syringealdehyde  lignin  vascular plant  angiosperms3. C37:3 alkenone  coccolith  marine 4. stable carbon isotopes trace carbon atoms through diets and up food webs Record time/maturation histories: 1.14C ticks away after removal from its atmospheric source 2.heating causes amino acids to racemize over time (L- alanine Æ D-alanine)

6. Record past events and processes histories (often cumulatively) white-rot fungal degradation of lignin increases the yield of vanillic acid versus vanillin from CuO oxidation of the wood remains  heating scrambles natural to unnatural structures  herbivory clips phytol tail off of chlorophyll molecule passage up through food webs fractionates nitrogen and carbon

7. Record paleoenvironmental conditions 1.temperature  number of C37:3 alkenone double bonds increases with decreasing water temperature 2.paleooxicity  reflected by some pigments and lipids 3.paleosalinity  reflected by some lipids

8. Organic information comes in useful “shells” 1. optical, microscopic, isotopic and bulk chemical (NMR, IR, CHN) characterizations are typically the most representative, but have limited information potential 2. molecular (biomarker) characterizations carry a wealth of information (including isotopic), but can often be unrepresentative of bulk organic mixtures which contain many organic components with different: a. sources b. physical forms c. ages d. reaction histories

9. The Major Biochemicals

10. General Rules for major biochemicals (beware of exceptions) • Macromolecules that are highly reactive within living cells also tend to be reactive outside the cell. EXAMPLE: structural cellular components (proteins, lipids, etc.) turn over in the cell more slowly than muscle, energy storage lipids and carbohydrates or enzymes, and are more likely to be detected in natural samples EXCEPTION: unusual structural characteristics or environmental circumstances can reduce the reactivity of molecules (DNA fossilized in bone). • The reactivity of organic matter depends greatly on the environment. Redox conditions, presence/absence of a mineral phase (biogenic or sedimentary) or light, and temperature can all dramatically change microbial reactivities. EXAMPLE: preservation of DNA requires isolation of material from a microbial community and also from oxygen (DNA in amber)

11. DNA/RNA (avg comp: C10H12N4O4) Deoxyribonucleic acid and ribonucleic acid The greatest potential as a biomarker, but usually poorly preserved in the environment • Structural unit is composed of a 5-C sugar (ribose), a nitrogen-rich cyclic base, and a molecule of phosphoric acid. The name of the compound is derived from its base (either a pyridine or purine ring). • DNA: genetic blueprint of the organism; machinery of evolution • RNA: messenger and transfer. RNA turns over rapidly between monomeric and polymeric form. It is recycled through the cell and is constantly being replaced.

12. adenosine guanosine uridine cytosine Strengths of DNA/RNA as geochemical tools: Ultimate biomarker Nitrogen-rich, but not in amide form, thus different from proteins Brings the biochemist into the geochemistry realm Weaknesses: poorly preserved except under extreme conditions Brings the biochemist into the geochemistry realm

13. CARBOHYDRATES Cx(H2O)y Most abundant biochemical class on earth due to cellulose and chitin A. Monomer (sugar) • most sugars have 5 (pentose) or 6 (hexose) carbon atoms • generally, carbohydrates are highly soluble in water and insoluble in non-polar solvents, and tend to decompose (caramelize) rather than melt • have a carbonyl group (HC=O) present as either an aldehyde (terminal) or a ketone (within the chain) • usually drawn as open chains but actually found as O-linked rings • ‘simple sugars’ usually include di- and tri-saccharides because they are sweet (table sugar = glucose-fructose) B. Diversity: there are many carbohydrates • chitin (n-acetyl glucosamine polymer) is unique to arthropods and fungi • bacteria make hundreds of unique deoxy, acidic, basic and O-methyl sugars

14. Lactose is found exclusively in themilk of mammals and consists of galactose and glucose in a b-(1,4) glycosidic bond.  C. Common Carbohydrates # Category Examples Carb Name ons 3 Triose glyceraldehyde, dihydroxyaceto ne 4 Tetrose erythrose 5 Pentose ribose, ribulose, xylulose 6 Hexose glucose, galactose, mannose, fructose 7 Heptose sedoheptulose . Polymer formation: dehydration to form etherlinkages. Sucrose is composed of glucose andfructose through an a-(1,2)b -glycosidic bond. 

15. Polysaccharides: the most abundant polysaccharides are cellulose, chitin, amylose, hemicellulose and pectin. With the exception of hemicellulose and pectin, all are homopolysaccharides (one sugar, usually glucose) linked together with chain lengths of ~1000-2000 units. Utility of carbohydrates as geochemical tools: • very abundant in living organisms • great deal of variability in the types of sugars made by microorganisms • optically active • Weaknesses of carbohydrates as geochemical tools: • Difficult to analyze; many different types ofcarbohydrates require multiple techniques • Instability of carbohydrates under many hydrolysis conditions adds to analytical difficulty • Wide variety of degradation rates makes bulkcarbohydrate measurements difficult to interpret

16. AMINO ACIDS C3.75H6N1O1.5 Forms: – Twenty common amino acids used to synthesize proteins. – More than 150 other amino acids are either used in cells for special purposes (taurine is used as an osmolyte in bivalves), as precursors to protein amino acids, created during certain typs of degradation sequences, or created abiotically. – Amino acid functional groups show great diversity: R=H for glycine but can contain rings (phenylalanine), sulfur (methionine), etc. – All amino acids (except glycine) contain 1 or more chiral carbons – Amino acids are zwitterions, having both basic and acidic groups. This leads to high solubility in water, low volatility and a tendency to decompose before melting • Polymer Formation: dehydrogenation reaction forms peptide bond (amide linkage) analogous to the glycosidic linkages in polysaccharides • Protein functions are varied and include enzymes, storage proteins, transport proteins, antibodies, toxins, hormones and structural proteins.

17. Strengths of amino acids as geochemical tools: – Structural diversity reveals a wide variety of processes and reactions – Reactions of epimerization and racemization provide an organic ‘clock’ reflecting temperature and time history – Sampling methodologies reasonably well worked out – Degradation of amino acids typically leads to a characteristic buildup of non-protein amino acids, yielding a relative index of degradation “freshness” – Some amino acids and proteins could provide source information • Weaknesses of amino acids as geochemical tools: – Little source information: nearly all organisms look similar in bulk amino acid composition – AA’s are typically quite reactive and can be rapidly lost from environmental samples

19. LIPIDS although certain characteristics are common across the compound class, lipids are very heterogeneous. • Lipids are operationally defined as being extractable by a nonpolar solvent • Common lipids: glycerides, waxes (including cutin and suberin), hydrocarbons, terpenoids (sterols, phytols, carotenoids), plus common heteromolecules (lipopolysaccharides, phospholipids, lipoproteins). • Cores are ‘polymethylenic’ (chains constructed from acetate units) or ‘polyisoprenoid’ ( chains or rings constructed of isoprene units) 2 general categories of lipids are saponifiable and nonsaponifiable (saponic = ‘soap producing”) based on presence of ester linkages

20. saponic lipids are composed of fatty acids and alcohols, which are released as monomers in boiling base. An example is palmitic acid (e.g. Palmolive), the most common fatty acid. All fatty acids contain a terminal carboxyl group. All fatty alcohols contain a terminal alcohol group. General characteristics: low solubility, melt rather than decompose,highly surface active, Saturated = full of hydrogen, no double bonds • Forming the lipid bond: • In general, fatty acids are linked to a • polyalcohol glycerol by ester linkages. • ii. Glycerides: 1,2 or all 3 of the alcohols on • the glycerol can link with a fatty acid to form • a glyceride. Chain length: ~C12-C36

21. Forming the lipid bond 2: • All archaea and some specific kinds of thermophilic bacteria • synthesis glycerol ethers as opposed to glycerol esters. • In archaea the chains are isoprenoidal while they are • polymethyleneic in bacteria

22. In general, animal fats are saturated, plant fats are unsaturated. For a given chain length, unsaturation leads to a lower melting point (lard is solid and canola oil is liquid at room temperature). In plants, the major fats are C18 (mono-, di- or tri-unsaturated). • Fatty acids are of even carbon number because they form from acetyl units H3C-CO-. • Function in cell: energy storage. For a given weight of compound, lipids yield ~2X more energy than carbohydrate. Why?

23. Waxes: an ester bond between a fatty acid and a fatty alcohol produces a wax ie wax ester. ‘Wax’ also refers to high MW hydrocarbons • Primary use in plants and insects as a protective coating and also as energy storage • Average chain length C24-C28 each end for a total of C48-C52 • some waxes contain ketones, branched alkanes, or aldehydes. When they contain alkanes, the number of carbon atoms becomes odd because the alkanes are formed by decarboxylation of fatty acids More on lipids…………

24. Terpenoids: highly diverse in structure and purpose, but all are divisible into isoprenoid units (± a couple of carbons). Monoterpenes (C10 = 2 isoprene units) are usually volatile, used as pheromones and stimulants (menthol, chrysanthemic acid) Sesquiterpines (3 units): oils and antibiotics Diterpenoids (4 units): Phytol side chain in chlorophyll. Most diterpenes are di- or tri-cyclic. Examples include gibberellins, abietic acid, vitamin A. testosterone

25. Triterpenoids (6 units) are all derived from squalene. Can be acyclic, tetra- or penta-cyclic. The most common tetracyclic ones are known as steroids. Major precursors to biomarker hydrocarbons in petroleum. steroids: tetracyclic triterpenoids used to provide rigidity in group, 1 double bond in the primary ring and a Pentacyclic triterpenoids are divisible into 4 classes: chain off the D-ring. oleananes, ursanes, lupines and hopanes. The first three are resins in vascular plants and they all have E-rings with 6 carbons. Hopanes are predominant in bacteria and have E-rings with 5 carbons.

26. Tetraterpenoids (8 units) usually form chains, important components of thic group include carotenoid pigments. Highly unsaturated, cyclicized at each end. Found in almost all organisms. Specific distributions of the carotenoids are characteristic of different organisms, especially photosynthetic organisms.

27. Characteristics of Biological MarkerCompounds – Optical Isomerism CCH3HCOOHCH3CHHOOCH2NNH2L alanineD alanine The D- and L-enantiomers of alanine are mirror image structures that cannot be superimposed. They can be distinguished by the direction in which they rotate a beam of mixtures and do not rotate the polarised light because the effect of the D-enantiomer is exactly cancelled by its L-counterpart. Unequal mixtures are said to be optically active and a compound comprising 100% of the D-enantiomer gives the maximum rotation clockwise or to the right while 100% of the L-enantiomer results in full anticlockwise (to the left) rotation. Most terrestrial organisms exclusively synthesise and use a-amino acids in the L-form.

28. Characteristics of Biological Marker Compounds – Limited # of Stereoisomers Stereoisomerism in tartaric acid. B and C are enantiomers. A, B and A, C are pairs of diastereomers.

29. Characteristics of Biological MarkerCompounds – Limited Stereoisomers Structure of cholesterol with its eight asymmetric carbon atoms identified with their position n umber. Theoretically, this compound could exist in as many as 256 (28) possible stereoisomers and yet biosynthesis produces only the one illustrated (Peters and Moldowan, 1993).

30. Characteristics of Biological Marker Compounds – Limited Structural Isomers Structures of a group of plant-derived C10 natural products (monoterpenes). The diphosphate ester of geraniol, itself formed by dimerization of D3isopentenyldiphosphate, is the biochemical precursor of the other structures. Limonene, myrcene and a-pinene are just three of the biologically-generated constitutional isomers with a molecular formula C10H16. There would be many hundreds of possibilities for non-natural isomers with the same formula.

31. Characteristics of Biological Marker Compounds –Repeating Sub-units (eg C2 or C5) Structure of the diterpenoid phytol composed of four head-tail linked C5 ('isoprene') units. Also note that phytol has two sites of asymmetry and a double bond each of which could deliver additional isomers if they were produced in other than natural circumstances. Phytol occurs uniquely as the E-3, 7R, 11R, 15-tetramethylhexadecene-2-enol structure

32. Characteristics of Biological Marker Compounds –Biologically defined Structures Structures of some regular, irregular and cyclic C20 isoprenoid (diterpenoid) hydrocarbons that have been identified in bitumen and which illustrate a variety of biosynthetic patterns based on repeating C5 dodecane are irregularly branched compounds while phytane, labdane and kaurane are constructed from four head-tail linked isoprene units. These compounds also illustrate how different structures can be diagnostic for specific physiologies (phytane for photosynthesis; crocetane for methanotrophy) or specific organisms (2,6,10-trimethyl-7- (3- methylbutyl)-dodecane for diatoms; labdane and kaurane for conifers).

33. Characteristics of Biological Marker Compounds – Systematic Isotopic Ordering at Molecular andIntramolecular Levels Cleavage of this bond in pyruvate induces fractionation • An important consequence of • the pyruvate to acetate • isotopic fractionation is • ‘lightness’ of lipids Alternate carbons derived from acetate carboxyl down acetogenic lipidbackbones are light. In general lipids are also light, but not as light.

34. DIAGNOSTIC MOLECULES MODERN & FOSSIL FORMS dinosterol dinosterane modern dinoflagellates Triassic-Recent

35. DIAGNOSTIC MOLECULES MODERN & FOSSIL FORMS C34 botryococcene B-raceof B. braunii C34 botryococcane lacustrine sediments Cenozoic-Recent

36. DIAGNOSTIC MOLECULES BACTERIAL LIPIDS REFLECT PROCESSES DIAGNOSTIC FOR PALAEOENVIRONMENT EUKARYOTE LIPIDS STRUCTURAL VARIETY DIAGNOSTIC FOR ORGANISMS & AGE

37. DIAGNOSTIC MOLECULESMODERN & FOSSIL FORMS oleanane β-amyrin ubiquitous in sediments modern angiosperms L. Cretaceous-Recent

38. DIAGNOSTIC PIGMENTS isorenierateneChloro bium carotenoid aryl isoprenoidsanoxia in photic zone