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
Hayes 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, 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. 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 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

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. C. Common Carbohydrates
# Category Examples
Carb Name
ons
Triose glyceraldehyde,
dihydroxyaceto ne
Tetrose erythrose
Pentose ribose, ribulose,
xylulose
Hexose glucose, galactose,
mannose,
fructose
Heptose 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 the
exception 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 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
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. 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 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 and Intramolecular 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-race of B. braunii
C34 botryococcane
lacustrine sediments
Cenozoic-Recent

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

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

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