Presentation on theme: "Molecular Fossils : Biomarkers"— Presentation transcript:
1 Molecular Fossils : Biomarkers Term ‘Biomarker’ also used inrelation to health of ecosystems andhumans Prognostic and PredictiveIndicators
2 Readings Hinrichs et al., Nature 1999 Thiel et al., GCA 1999 – Both papers on linking biomarkers to AOM– Potential examination materialFogel and Cifuentes 1994, Isotopic (esp.13C)fractionation during primary productionHayes 2002, 13C and 2H-fractionation during biosynthesis– For the really committed!!!!!!; mostly not examinable
3 Why is nonliving natural organic matter important? Dimensions of Organic Geochemistry Based 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, drugsWhy is nonliving natural organic matter important?food, fuel, structural materials, drugs plays an important role in globalcycles of C, O, N, P, S, metals etc affects soil fertility and structure,redox chemistry, photochemistry, light transmission record ofenvironmental conditions, history and reactions (molecular fossils)
4 Data richnesslarge number of information units = isotopes, atoms, molecules, stereochemistrya. 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 Record time/maturation histories: Organic matter traces biological and geographic sources “biomarkers”: 1. vanillin lignin vascular plants terrigenous origin 2. syringealdehyde lignin vascular plant angiosperms 3. C37:3 alkenone coccolith marine 4. stable carbon isotopes trace carbon atoms through diets and up food websRecord time/maturation histories:1.14C ticks away after removal from its atmospheric source2.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 ligninincreases the yield of vanillic acidversus vanillin from CuO oxidation ofthe wood remains heating scrambles natural to unnaturalstructures herbivory clips phytol tail off of chlorophyllmoleculepassage up through food webs fractionates nitrogen and carbon
7 Record paleoenvironmental conditions 1.temperature number ofC37:3 alkenone double bondsincreases with decreasing watertemperature2.paleooxicity reflected bysome pigments and lipids3.paleosalinity reflected bysome 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 potential2. 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. sourcesb. physical formsc. agesd. reaction histories
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 samplesEXCEPTION: 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 acidThe greatest potential as a biomarker, but usually poorly preserved in the environmentStructural 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 evolutionRNA: 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 biomarkerNitrogen-rich, but not in amide form, thus different fromproteinsBrings the biochemist into the geochemistry realmWeaknesses:poorly preserved except under extreme conditions
13 CARBOHYDRATES Cx(H2O)y Most abundant biochemical class on earth due to cellulose and chitinA. Monomer (sugar)most sugars have 5 (pentose) or 6 (hexose) carbon atomsgenerally, carbohydrates are highly soluble in water and insoluble in non-polar solvents, and tend to decompose (caramelize) rather than melthave 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 carbohydrateschitin (n-acetyl glucosamine polymer) is unique to arthropods and fungibacteria make hundreds of unique deoxy, acidic, basic and O-methyl sugars
14 C. Common Carbohydrates # Category ExamplesCarb NameonsTriose glyceraldehyde,dihydroxyacetoneTetrose erythrosePentose ribose, ribulose,xyluloseHexose glucose, galactose,mannose,fructoseHeptose sedoheptulose. Polymer formation: dehydration to form etherlinkages. Sucrose is composed of glucose andfructose through an a-(1,2)b -glycosidic bond. Lactose is found exclusively in the milk of mammals and consists of galactose and glucose in a b-(1,4) glycosidic bond.
15 Polysaccharides: the most abundant polysaccharides are cellulose, chitin, amylose, hemicellulose and pectin. With theexception of hemicellulose and pectin, all are homopolysaccharides(one sugar, usually glucose) linked together with chain lengths of~ units.Utility of carbohydrates as geochemical tools:very abundant in living organismsgreat deal of variability in the types of sugars made by microorganismsoptically activeWeaknesses of carbohydrates as geochemical tools:Difficult to analyze; many different types ofcarbohydrates require multiple techniquesInstability of carbohydrates under many hydrolysis conditions adds to analytical difficultyWide 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 forspecial purposes (taurine is used as an osmolyte in bivalves), asprecursors to protein amino acids, created during certain typs ofdegradation sequences, or created abiotically.– Amino acid functional groups show great diversity: R=H for glycinebut 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 tendencyto decompose before meltingPolymer Formation: dehydrogenation reaction forms peptide bond (amide linkage) analogous to the glycosidic linkages in polysaccharidesProtein 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 processesand 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 characteristicbuildup of non-protein amino acids, yielding a relative index ofdegradation “freshness”– Some amino acids and proteins could provide source informationWeaknesses of amino acids as geochemical tools:– Little source information: nearly all organisms look similar in bulkamino acid composition– AA’s are typically quite reactive and can be rapidly lost fromenvironmental 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 solventCommon lipids: glycerides, waxes (including cutin and suberin), hydrocarbons, terpenoids (sterols, phytols, carotenoids), plus common heteromolecules (lipopolysaccharides, phospholipids, lipoproteins).Cores are ‘polymethylenic’ (chains constructed fromacetate units) or ‘polyisoprenoid’ ( chains or ringsconstructed 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 bondsForming the lipid bond:In general, fatty acids are linked to apolyalcohol glycerol by ester linkages.ii. Glycerides: 1,2 or all 3 of the alcohols onthe glycerol can link with a fatty acid to forma glyceride. Chain length: ~C12-C36
21 Forming the lipid bond 2: All archaea and some specific kinds of thermophilic bacteriasynthesis glycerol ethers as opposed to glycerol esters.In archaea the chains are isoprenoidal while they arepolymethyleneic in bacteria
22 In general, animal fats are saturated, plant fats are unsaturated 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 hydrocarbonsPrimary use in plants and insects as a protective coating and also as energy storageAverage chain length C24-C28 each end for a total of C48-C52some 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 acidsMore on lipids…………
24 Terpenoids: highly diverse in structure and purpose, but all are divisible into isoprenoidunits (± 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 antibioticsDiterpenoids (4 units): Phytol side chain in chlorophyll. Most diterpenes are di- or tri-cyclic. Examples include gibberellins, abietic acid, vitamin A.testosterone
25 steroids: tetracyclic triterpenoids used to provide rigidity in 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 ingroup, 1 double bond in the primary ring and aPentacyclic triterpenoids are divisible into 4 classes:chain off the D-ring. oleananes, ursanes, lupines andhopanes. The first three are resins in vascular plants andthey all have E-rings with 6 carbons. Hopanes arepredominant 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 alanineThe D- and L-enantiomers of alanine are mirror image structures that cannot besuperimposed. They can be distinguished by the direction in which they rotate a beam ofmixtures and do not rotate the polarised light because the effect of the D-enantiomer isexactly cancelled by its L-counterpart. Unequal mixtures are said to be optically activeand a compound comprising 100% of the D-enantiomer gives the maximum rotationclockwise or to the right while 100% of the L-enantiomer results in full anticlockwise (tothe left) rotation. Most terrestrial organisms exclusively synthesise and use a-amino acidsin 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 atomsidentified with their position n umber. Theoretically, this compoundcould exist in as many as 256 (28) possible stereoisomers and yetbiosynthesis 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 bydimerization of D3isopentenyldiphosphate, is the biochemical precursorof the other structures. Limonene, myrcene and a-pinene are just threeof the biologically-generated constitutional isomers with a molecularformula C10H16. There would be many hundreds of possibilities fornon-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 asymmetryand a double bond each of which could deliver additional isomers ifthey were produced in other than natural circumstances. Phytol occursuniquely as the E-3, 7R, 11R, 15-tetramethylhexadecene-2-enolstructure
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 andwhich illustrate a variety of biosynthetic patterns based on repeating C5dodecane are irregularly branched compounds while phytane, labdaneand kaurane are constructed from four head-tail linked isoprene units.These compounds also illustrate how different structures can bediagnostic 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 forconifers).
33 Characteristics of Biological Marker Compounds – Systematic Isotopic Ordering at Molecular and Intramolecular LevelsCleavage of thisbond in pyruvateinduces fractionationAn important consequence ofthe pyruvate to acetateisotopic fractionation is‘lightness’ of lipidsAlternate carbons derived from acetate carboxyldown acetogenic lipidbackbones are light. Ingeneral lipids are also light, but not as light.
34 DIAGNOSTIC MOLECULES MODERN & FOSSIL FORMS dinosterol dinosteranemodern dinoflagellates Triassic-Recent
35 DIAGNOSTIC MOLECULES MODERN & FOSSIL FORMS C34 botryococceneB-race of B. brauniiC34 botryococcanelacustrine sedimentsCenozoic-Recent