Molecular Biogeoch emiststry Wk. 3

• Hopanoids and other cyclic terpenoids – Structures, biosynthesis, diagenesis – Hopanoid hydrocarbons; stereochemistry vs maturity – Hopanoids as process and environment indicators Hopanoids

• First recognised as a class of

C30 pentacyclilic triitterpenes found in ferns, mosses and dammar resinsresins These images have been removed due to copyright restrictions. • ‘Hopane’ named after the Dipterocarp plant genus Hopea, itself after botanist John Hope

• Biosynthetic kinship to sterols, tetrahymanol & oleanoids, via squalene recognised in 60’s

© Gaines, Eglinton & Rullkötter Evolution of Hoppyane & Sterol Bioynthesis

BHP Squalene Dippploptene o2 BACTERIA

Squalene epoxide O

o2 EUCARYA

HO HO C24 substitution Lanosterol Cholesterol by algae some bacteria - Methylococcus Mycobacteria, Myxobacteria Structure and Function of a Squalene Cyclase K. Ulrich Wendt, Karl Poralla, Georg E. Schulz * The crystal structure of squalene-hopene cyclase from Alicyclobacillus acidocaldarius was determined at 2.9 angstrom resolution. The mechanism and sequence of this cyclase are closely related to those of 2,3-oxidosqualene cyclases that catalyze the cyclization step in cholesterol biosynthesis. The structure reveals a membrane protein with membrane-binding characteristics similar ttoo tthosehose of prostaglandin -H 2 synthase, thethe only other reported protein of this type. The active site of the enzyme is located in a large central cavity that is of suitable size to bind squalene in its required conformation and that is lined by aromatic residues. The structure supports a mechanism in which the acid starting the reaction by protonating a carbon-carbon double bond is an aspartate that is coupled to a histidine. Numerous surface helices are connected by characteristic QW-motifs (Q is glutamine and W is tryptophan) that tighten the protein structure, possibly for absorbing the reaction energy without structural damage. http://biol.lancs.ac.uk/StuWork/BIOS316/Bios31699/squacyc/316-2.htm#Function SQUALENE 3 1

B1:H B2

HOPENE 22

29

The proposed reaction steps in squalene-hopene cyclases involving carbocationic intermediates. The general acid B1:H protonates (H) squalene at C3, whereas the general base B2 deprotonates at C29 of the hopenyl cation. In a side reaction, the cation is hydroxylated forming hopan-22-ol (i.e. diplopterol)

Figure by MIT OpenCourseWare. This image has been removed due to copyright restrictions.

Figure 5. The color-coded surface representations (30) with nonpolar (yellow), positive (blue), and negative (red) areas. (A) View similar to Fig. 2 but rotated around a vertical axis and sliced. The cutting plane (checked) opens the large internal cavity with thethe bou nd inhibitor LDAO. TThehe nonpolar channel runs ttoo tthehe left, opening into a nonpolar plateau. The channel constriction (C) appears closed, but it is mobile enough to be readily opened. At the upper left, hopane (two views) is shown at scale. (B ) View similar to Fig. 2 directly onto the 1600 Å2 nonpolar plateau with the channel entrance (E) at its center and two nonpolar side chains pointing to the outside. This is the only large nonpolar region on the surface Discovery of Geohopanoids

• Hopane identified in Green River Shale by Burlingame, Haug, Belksky & Calvin, PNAS, 1965.

• C27-C31 Triterpanes in optically active petroleum distillates, Hills & Whitehead, Nature 1966

• Homohopane in Green River, Ensminger Maxwell (Bristol & Strasbourg, ‘72)

• Extended hopane series to C35 ubiquitous in the geosphere incl. petroleum, soils and diverse sediments Ensminger, van Dorsselaer, Spyckerelle, Albrecht, Ourisson (Strasbourg) and Eglinton, Maxwell, Kimble, Philp & Brooks (Bristol) 1973-4

Hills & Whitehead (BP) speculated there was a C35 precursor Hopanoids in Bacteria & Rocks?

• Diploptene (C30) identified in bacteria & cyanonbacteria, de Rosa (1971) incl. Methylococcus capsulatus , Bird et al.,,((1971)

•ββ-hopane and ββ-homohopane in Bacillus acidocaldarius, de Rosa 1973

• Fossil hopanoids exhibit αβ, βα and ββ stereochemistry with 22S+R; ββ and βα recognized as less stable than αβ

• C30 C-3 oxygenated triterpane alcohols and ketones in Messel, but not equivalent C30 hydrocarbons sug gggest many ho pane hydrocarbons entered as 3-desoxy components

• Mystery ‘almost ’ solved when Forster, Biemann et al characterized a C 35 tetrahydroxy triterpenoid in Acetobacter xylinum (1973) Hopanoids

Biochem. J. (1973) 135, 133-143 133 Printed in Great Britain

The Structure of Novel C35 Pentacyclic Terpenes from Acetobacter xylinum By HANS J FŎRSTER * and KLAUS BEIMANN Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Mass. 02139, U.S.A.

And W.GEOFFREY HAIGH Biomedical Research Laboratory, Dow Corning Corporation, Midland, Mich, 48640, U.S.A.

And NEIL. H.TATTRIE and J.ROSS COLVIN, Division of Biological Sciences, National Research Council of Canada, Ottawa K1A OR6, Canada (Received 14 February 1973) Patterns of Geohopanoids in GC-MS

Geochemists figured that the absence of C28 homologue and pairs of peaks from C 31 -C35 are iinformativenformative about side -chain structure Hopanoids

Courtesy Elsevier, Inc., http://www.sciencedirect.com. Used with permission. Hopanoids

Courtesy Elsevier, Inc., http://www.sciencedirect.com. Used with permission. Hopanoids

A novel C35 terpene and its monounsaturated analogue were isolated from cultures of Acetobacter xylinum, together with traces of their C36 homologues. These substances were found to be hopane derivatives substituted by a five-carbon chain bearing ffoo ur vicinal h ydroxyl groups . For tthehe parent h ydrocarbon the term bacteriohopane is proposed. The elucidation of the structures utilized high-resolution mass spectrometry of the terpenes, degradation to C32 hydrocarbons and detailed mmassass -spectrometric ccomparisonomparison of these wwithith C32 hydrocarbons synthesized from known pentacyclic triterpenes. High- resolution mass-spectral data of the terpenes are presented. N.m.r. data are in agreement with the proposedproposed structures, whichwhich are furtherfurther supported bbyy the isolation from the same organism of 22-hydroxyhopane and derivative hopene(s). Hopanoid quotes • The formation of the more stable αβ hopane epimers could constitute , in a given environment, a geochemical clock unless they happen to be still unrecognized constituents of living organisms …...... Ensminger et al., Advances in OG..1973 • The total amount of geohopanoids is estimated to be ~1012 tons and same order as total mass of organic carbon in all living organisms …...... Ourisson and Albrecht, Geohopanoids, the most abundant natural products on Earth, Acc Chem Res 1992 Hopanoids

This image has been removed due to copyright restrictions.

In: A.G. Douglas and J.R. Maxwell, Editors, Advances in Organic Geochemistry, Pergamon Press, Oxford (1980), pp. 229–237 Hopanoids & Thermal Maturity Structural Diversity of Hopanoids

Courtesy Elsevier, Inc., http://www.sciencedirect.com. Used with permission. Hopanoids and Physiology

Distribution of hopanoid triterpenes in prokaryotes

Rohmer, M. , Bouvier-Nave, P. , Ourisson, G. Ecole Nationale Superieure de Chimie de Mulhouse, Universite de Haute Alsace, 68093 Mulhouse Cedex, France

Present in 50% of some 100 strains across diverse taxa

In almost all cyanobacteria, obligate methylotrophs , purple non-sulfur bacteria and diverse chemoheterotrophs

Absent from purple sulfur bacteria, archaebacteria

Not detected in any anaerobe

Bacteriohopane tetrol most common Journal of General Microbiology Volume 130, Issue 5, 1984 1137-1150 Functional Role of Hopanoids?

Rohmer & OOurisson,urisson, 1971976

Rohmer et al., 1979

Kannenberg & Poralla , 1980

Many lines of evidence show an association of BHP with cellular membranes

Image courtesy of Michel Rohmer. Discoveryof Meth ylated Hopanoids

Acetobacter BHP hypothesized to be 3­ methylhopanoids on biosynthetic grounds Discovery of 2β-Me Hopanoids 2β-Me Hopanoids

2β by nmr & direct comparison with 2β-methyldiplopterol This image has been removed synthesized f rom 2222­ due to copyright restrictions. hydroxyhopan-3-one. 2α-Me Hopanoids

c. 5% methyldiplopterols 2α by 13 C nmr & direct comparison with 2α-methyldiplopterol synthesized f rom 2222- hdhydroxy hopan- 3-one.

Sloppy methylase? Any 2α-methyl in Nostoc or Rhodopseudomonas? Geological Occurrence of Methyl Hopanoids • Fossil methyl hopanes reported in sedimentary rocks and oils eg SEIFERT and MOLDOWAN, 1978; ALEXANDER et al., 1984; MCEVOY and GIGER, 1986; SUMMONS and POWELL, 1987 • Tentatively but erroneously thought to be 3-methyl hopanes based on the reports of 3-methylhopanoids in metthhylotrop hs • Complex mixtures precluded any spectroscopic analysis oth er than GC-MS • Synthetic approaches already developed by Rohmer but not applied to geo hopanoids Geological Occurrence of Methyl Hopanoids 100 68.46

% 71.28 71.77 71.09 454.45273.29 > 205.194 66.14 67.02 69.04 72.43 74.22 0 03072401 67.97 100 68.38 % 440.437 > 191.179 66.88 68.96 70.39 0 03072401 66.22 100 66.47

% 69.16 69.45 440.437 > 205.194 66.96 68.03 70.39 65.19 0 03072401 66.12 100 66.37 % 426.421 > 191.179 66.88 63. 998864.83 67.1167 .9977 0 03072401 66.14 100 66.39 64.09 % 66.72 63.98 64.25 426.421 > 205.194 60.00 62.81 67.99 68.40 0 03072401 63.98 100

% 63.74 412.406 > 191.179 61.76 62.63 64.13 66 .12 66.39 0 60.00 62.00 64.00 66.00 68.00 70.00 72.00 74.00 76.00 78.00 GC with MS-MS -> isomers of each individual homologue to be seen in separate chromatograms Geological Occurrence of Methyl Hopanoids

Comparison of 2α ­ methyl hopane and 3β ­ methyl hopane with This image has been removed fossil hydrocarbonshydrocarbons due to copyright restrictions. Sample was an immature Ordovician sediment with two series of methyl hopanes Geological Occurrence of Methyl Hopanoids

Relative retention times used to assign identity

to C27-C35 series’

Sample s an immature Ordovician kukersite

Sedidimen t ary methyl h opanes deri ved f rom methyl anal ogues of BBHPHP O2 -Diagnostic Bacteriohopanes ??

OH OH 19 21 22 32 35 diagenesis R 25 11 17 3 14 16 R OH R maturation 29 1 2 1 2 10 3 4 cyanobacteria cyanobacteria δ13C -20 to -35 ‰

OH OH 19 21 22 diagenesis 32 35 R 25 11 17 maturation 3 14 16 OH OH NH 29 2 1 2 10 3 4 methanotrophic bacteria methanotrophs δ13C -45 to -80 ‰

Jahnke et al., 1992, 1993; 1999; Summons & Jahnke, 1988, 1990; Summons et al., 1994, 1999 Isotopic Signature of 3-Methyl Hopanoids

pMMO sMMO

C-isotopic fractionation in biomass of M. capsulatus: varies as a function of substrate, growth stage, MMO type

Summons et al., GCA, 1994 Courtesy Elsevier, Inc., http://www.sciencedirect.com. Used with permission. Isotopic Signature of 3-Methylhopanoids

Biomass -triterpenoids ε ~ 36‰

Distribution and C-isotopic fractionation in hopanoids of M. capsulatus as function of growth stage Summons et al., GCA, 1994 Courtesy Elsevier, Inc., http://www.sciencedirect.com. Used with permission. Geological Occurrence of 3-Methylhopanoids

This image has been removed due to copyright restrictions. Please see Figure 7 in the above article. Hamersley Basin biomarker & isotopic records

Courtesy of National Academy of Sciences, U. S. A. Used with permission. Source: Eigenbrode et al (2008) National Academy of Sciences, USA. Eigenbrode et al, PNAS Copyright (c) 2008, National Academy of Sciences, U.S.A. 2008 Cyanobacterial Lipids

9 COOH OH phytol 11 COOH

C 16 -18 fatty acid and PUFA

n-heptadecane

7 β -carotene 7- & 8­ methylheptadecane 8 R OH 7 11

717,111- dimethylhep tadecane hopanoids & 2-methylhopanols Are 2-methylhopanes biomarkers for

cyanobacteria and O 2-photosynthesis?

• Specificity of 2-MeBHP for Cb? ~30% of Cb surveyed (Talbot et al, OG 2008) – Methylobacterium, N-fixing soil bacteria (Rhizobiales) – Rhodopseudomonas palustris (Rashby et al., 2007) – Commonly present in heterocystous cyanobacteria – Relatively abundant in a BBalticaltic SSeaea strain of Nodularia • What are the environmental occurrences? – Very abundant in hot spring mats; low levels in marine hypersaline Cb mats • What are the biological functions of BHP & methylated BHP?

– Membrane-associated in P luridum and Mc Bath; pCO2 control in Phormidium? • Geological occurrences?occurrences? Secular in shales; Always aatt OAEs • What is the biosynthetic pathway? – No unsaturated precursors; Radical SAM? See Paula’s talk • Are biosynthetic pathways conserved over geological time? Does a biomarker found today mean the same thing as one 500Myr old? 2-MeHopanes vs Lithology & Paleogeography

0.30

Carbonate Marl

020.255 Distal Shale

0.20 x x nde I ne

0.15 hopa l yy

2-Meth 0.10

0.05

0.00 -90 -70 -50 -30 -10 10 30 50 70 90 Paleolatitude 2-Methylhopane Index 0.10 0.20 030 2-Me . 30 0 BHP 200 - B iomarkers for 400 600 Sediment Age Cyanobacterial Photosynthesis? 800 1000 MY 1200 A 1400 1600 Distal Shale Marl Carbo n a t e 1800 2-Me BHP - Biomarkers for Frasnian-Famennian Cyanobacterial Photosynthesis ? 0.30 Carbonate Toarcian Marl Distal Shale

x x OAE Permian-Triassic Cretaceous OAEs

ne Inde 0.20 aa thylhop ee 2-M 0.10

0 100 200 300 400 500 600 Sediment Age MYA 2α-Methylhopane index 2.72-2.56 Ga samples from the Hamersley Province

Courtesy of National Academy of Sciences, U. S. A. Used with permission. Source: Eigenbrode et al (2008) National Academy of Sciences, USA. Eigenbrode et al, PNAS Copyright (c) 2008, National Academy of Sciences, U.S.A. 2008 Meishan Drilling Project 2004

c.120 samples Late Permian-Mid Triassic for 87 86 13δ 13δ 15δ bulk geochemistry: Sr/ Sr, TOC, carb, org, org and detailed lipid biomarkers

Meishan-1 core drilled near GSSP Jan 2004

This image has been removed This image has been removed due to copyright restrictions. due to copyright restrictions.

Multiple radiometric ages constrain pace; Ash in bed 25 = 251.4 ± 0.3 Ma, Bowring et al, 1998; Revised 252.6 ± 0.2 Ma Mundil, 2004; & 252.25 ± 0.06 Crowley, Bowring 2008 δ15N of Meishan Organic Matter

-ve -85 +ve z• PiPosititive values ((+3+3 to ++2) 2) in l ate -4 -2 0 2 4 Permian Beds 6 -24

δ15 -90 • Trend to zero or negative values of N 37-2 in latest Permian reflects depletion of 36-3 nitrate/nitrite pool driven by euxinic 35-1 35-2 -95 conditions

34-12 • Large swings in E. Triassic may reflect

-100 waxing and waning of euxinia depth/m 34-3 • Predominantly cyanobacterial primary 34-1 N fixation -105 30-1 26-2 b24 Peaks iinn 2 -MeHI >15% 23-4 -110 22-3 Peaks of aryl isoprenoids

-115 δ15N kerogen EARTHRISE

Certified Pesticide FREE

SPIRULINA

Blue-Green Algae Rich in Beta Carotene Essential Vitamins & Phytonutrients Biologically Grown in the USA

100 TABLETS Net Wt. 50 grams 2-Methylhopane Index for Sediments/Oils Barney Creek Fm #1698 αβ-hopane

412 ->191

2α(Me) -αβ- hopane 2α-MeH Index

= 2 α(Me )/2α (Me )+ αβ 426 ->205

1:00:00 1:04:00 Time Life’s History on Earth Cloud, Holland, Walker Paradigm Prokaryote World

Multi-cell Life 1

0.1 Humans

First First 0.01 Eukaryotes GOE Invertebrates (algae) atm) (( 2 O

P 0.001

Oldest ‘compelling’ 0.0001 fossil, stromatolite and C- isotopic evidence for life

0.00001 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 Time before Present (Ga) Molecular Biosignatures:

Real and Potential Biomarkers, Analytical Innovations, Meteorites & Old Rocks Topics

• What are useful criteria for biogenicity? How can we be sure of measuring the right thing in a sample on Mars or returned from Mars?

• Analytical methods for investigating molecular biosignatures in rocks from Earth & elsewhere

• New ‘technologies ’ in molecular biosignatures for organisms and ancient environments Hydropyrolysis & Strelley Pool Chert Assumptions

• Extra- terrestrial life will resemble earthly life – based on carbon chemistry operating in an aqueous environment - carbon is the only eelementlement that is sufficiently abundant , ubiquitous and chemically suited for life • It will process chemicals for carbon and energy, make copies of itself , be autonomous and evolve in concert with its environment • Biochemical pathways will operate aass above – comprise energy yielding and replication reactions – construct complex molecules from simple, universal precursors – evolve Abiotically produced organic materials

Organic acids and diacids, amino acids, hydroxy acids, alcohols, amines

• n- and branched-hydrocarbons incl. methane

• Aromatic hydrocarbons (PAH)

Message: Biological organic materials are patterned Building BlocksÆ 20 amino acids, 4 bases, 2 lipid types……. Intrinsic Characteristics (Patterns) of Terrestrial Molecular Biosignatures

• Enantiomeric excessexcess • Diastereoisomeric preference • Constitutional isomer preference • Repeattiing cons tittitutitiona lsu b-units or atomic ratios • Systematic isotopic ordering at molecular and intramolecular levels • Systematic distribution patterns or clusters (e.g. C-number, concentration, d13C) of structurally related compounds Enantiomers of Alanine

H H

CH3 H3C H2NC C NH2

COOH HOOC L-alanine D-alanine

L-amino acids predominate in biology L-amino acid XS in Murchison meteorite (Engel & Macko a-aa’s; Cronin & Pizzarello non-protein aa’s) Non-biological processes can yield enantiomeric excess Asymmetric catalysis and autocatalysis Soai & Sato: slight chiral excess propagated during autocatalytic syntheses Pizzarello and Weber: AA enantiomeric excess promotes asymmetry in aldol condensations of glycoaldehyde Stereoisomerism in Tartaric Acid

H COOH HOOC H H COOH OH H H C C C

C C C OH OH OH HOOC H HO COOH HOOC OH

A (meso) B C

B & C enantiomers A & B and A & C are diastereoisomers Life makes a limited number of all the possible di astereoisomers Stereoisomerism in Cholesterol

20

13 17 9 14

10 8 3 H H HO

28 stereoisomers possible f or chol esterol Biology (ie Eucarya) makes only one Studies of hydrocarbons as old as 2700 Myr show no deviation of sterane or hopane stereoisomer patterns; the fossils had the same precursors as exist today Information Preserved in Products of Diagenesis: The SSterolterol PPathwaysathways Unique sterol configuration of living organisms 8 sites of asymmetry 28 = 256 feasible isomers sterenes and steranes but only 1 in nature immature sediments αα (20R) R R 20 aa20R

o17 o rearranged steranes o o aromatic steranes 14

HO R

R R R o o o o

ββ (20S) ββ(20R) o αα(20S)

Complex sterane mixture in mature sediments & oil Constitutional Isomers

OH

OH OH

(S)-Ipsenol Geraniol Nerol Myrcene Pine beetle pheromone

Choleoptera: Scolytidae C10 compounds (monoterpenes) - life makes a specific subset of all the possibilities

O

(1S)-(-)- β- Pinene O

OH

(1R,2S,5R)-(-)-Menthol (S)-(-)-Limonene Cineol (eucalyptol) O

(1R)-(+)-Camphor (1R)-(+)-α-Pinene Constitutional Isomers

α β γ δ ε ζ HOOC - C - C - C - C - C - C

2 or OH X = NH Small molecules identified in the Murchison Meteorite tend to occur with the maximum number of possible theoretical isomers e. g. monoamino monocarboxylicmonocarboxylic acids and monohydroxy, monocarboxylic acids (Cronin et al. 1993) Constitutional Isomers

α β γ δ ε ζ HHOOCOOCCCCCCC - C - C - C - C - C - C

XNX = NHH2 or OH

C-atoms α β γ δ ε ζ unknown 2 1, 1, 1 ------3 1, 1, 1 1, 1, 1 ------4 2, 2, 2 2, 2, 2 1, 1, 1 ------5 3, 3, 3 6, 6, 3 3, 3, 3 1, 1, 0 -- -- 1 6 8 , 8 , 8 12 , 3, 1 11 , 4 , 0 4 , 2 ,0 1 , 1 , 0 -- 2 7 18, 18, 12 29, 0, 0 29, 0, 0 20, 0, 0 5, 0, 0 1, 0, 0 2 Acetogenic Lipids & Polyisoprenoids

11 7 3 OH Life makes a limited number of all the possible constitutional isomers because it : - has evolved ‘universal’ biochemical pathways - constructs macromolecules from small, common precursors (eg 20 amino acids iinn protein, 4 bases of DNA)

Æ• preference for certain carbon numbers (clusters) & systematic isotopic ordering within & between molecules Clusters of compounds C 15 - example of a leaf wax ‘cluster ’ ‘d‘oddd over even’ isoprenoids C20 C29-C33 ‘cluster’ of ‘cluster’ n-alkanes

H COOHisoprenoids HOOC H H COOH OH H H C C C C C C OH OH OH HOOC H HO COOH HOOC OH

B C

10 20 30 40 50 60 70 min Clustering is a consequence of universal biochemical pathways Clusters of Compounds - example of a sediment ‘odd over even’

C29-C33 ‘cluster’ of n -alkanes C20 ‘cluster’ of diterpenes

C30 and C35 ‘clusters’

25.00 30.00 35.00 40.00 45.00 50.00 55.00 NB clustering is ‘blurred’ by mixed inputs, diagenesis, catagenesis Isotopic Ordering PlPolyisoprenoiidd lliiipidds

This image has been removed due to copyright restrictions . Polymethylenic = acetogenic lipids

Isotopic ordering is a consequence of the universality of biochemical pathways

Hayes J. M. (2002) Fractionation of the isotopes of carbon and hydrogen in biosynthetic processes. In: Stable Isotopic Geochemistry, Valley J. W. and Cole D.R. (eds.) Reviews in Mineralogy and Geochemistry.

Acetogenic Lipids

n-Alkyl lipids are essentially polymers of acetate – acetogenic lipids

‘heavy’ O CH3 O CH3 ● C O ● C SCoA O + CO 2 ‘light’ + CO2

From Hayes, 2002 Acetate Methyl-C and Carboxyl-C are isotopically distinct and determined by its metabolic source and the profound isotope effect of pyruvate dehy drogenase Origin of C-Atoms in Polyisoprenoids & Consequent ItIsotopic Orderi ng

a, c = MVA b, d = MEP IsoprenoidsObserving thisshould at natural encode abundance either of thesepresently ordering a challenge patterns n-Alkane Carbon Isotopes in Oils

12 14 16 18 20 22 24 26 28 30 32 -33.00

-34.00

-35.00

Suwaihat-8 Unk Str Sakhiya-1 A3C Qashoob-1 A2C δ δ -36 .00 Budour NE-1 A4C 13 Hudaira-1 A5C Budour NE-2 A1C Dhahaban East-2 A1C -37.00

-38.00

-39.00 Carbon Number Murchison

1.2 202 Pyrene/ Fluoranthene 178 101.0 Phenanthrene/ Dominance of Anthracene 0.8 2-4 ring parent PAH ity

ss otherwise similar n 0.6 128 to ALH84001 Naphthalene l Inte

gna 0.4

Si Chrysene/ Benzo(a)anthracene 0.2 Benzo(a)pyrene/ Benzo( b/k) fluoranthene -0.0

-0.2 50 100 150 200 250 300 350 Mass (amu) Feb 1, 99 Murchison

111.1

1.0

0.9 Alk yl ati on drops exponenti all y 0.8 ie no ‘clusters’ ce 0.7 an dd 0.6 bun A

e 0.5 iv tt a 0.4 Rel 0.3

0.2

0.1

000.0 -10 1 2 3 4 5 6 Number of Carbons attached to Phenanthrene Feb 1, 99 PAH Proposed as molecular fossils ?

PHENANTHRENE 1-METHYL- 1,7-DIMETHYL- 178 PHENANTHRENE 192 PHENANTHRENE 206

1,2,5-TRIMETHYL- RETENE PHYLLOCLADANE NAPHTHALENE 156 234

R R3

R4

R1 R2 δ13 C Murchison oorganicrganic ccompoundsompounds 40 aromatic hydrocarbons aliphatic hydrocarbons 30 carboxylic acids sulphonic acids 20 amino acids 10 δ13 C 0 ‰PDB -10 Sephton et al., 1998 Cooper etet alal., 1997 -20 Gilmour and Pillinger 1994 Engel et al., 1990 -30 Pizzarello et al., 1991 Yuen et al., 1984 -40 0 5 10 15 20 25 carbon number Compilation courtesy of Mark Sephton Topics

• What are usefuluseful criteria forfor biogenicity? How can we be sure of measuring the right thing in a sample on Mars or returned from Mars? Deliberations of the 2000 NASA Biomarker Taskforce – molecular biosignatures •Analytical methods for investigating molecular biosignatures in rocks from Earth & elsewhere GC2-TOFMS • New ‘technologies’ in molecular biosignatures for organisms and ancient environments Hydropyrolysis & Strelllley Pool ChChert OMR017 Buah Fm.

Nelson, Reddy, Freysinger + MIT, unpublished Loop-Jet Modulator

Cold Jet Injector Detector

Hot Jet

1st Column 2nd nonpolar Delay loop Column polar

Blank 0.8 meters Rtx-1 6.5 meters BPX-50 0.8 meters

Figure by MIT OpenCourseWare. b OMR017 a c Buah Fm d f e

g h i j l k m

a. 17α(H),21β(H)-30-norhopane b. 17α(H),21β(H)-hopane c. 17α(H),21β(H)-29-homohopane 22S d. 17α(H),21β(H)-29-homohopane 22R e. gammacerane f. 17α(()H),,21 β(()H)-29-bishomoho pane 22S g. 17α(H),21β(H)-29-bishomohopane 22R h. 17α(H),21β(H)-29-trishomohopane 22S i. 17α(H),21β(H)-29-trishomohopane 22R j. 17α(H),21β(H)-29-tetrakishomohopane 22S k. 17α(H), 2211β(H)- 29 -tetrakishomohopane 22R l. 17α(H),21β(H)-29-pentakishomohopane 22S m. 17α(H),21β(H)-29-pentakishomohopane 22S

Nelson, Reddy, Freysinger + MIT, unpu 20 Carbons 19 Carbons 18 Carbons

17 Carbons

16 Carbons

15 Carbons OMR011 - Thu l eilil at Fm 14 Carbons Nelson, Reddy, Freysinger + MIT, unp Cyclohexane + n-C12 55

82

2.5

67

88 89 90 91

97 252 M+ 252 = C18

50 100 150 200 250 300

Nelson, Reddy, Freysinger + MIT, unpublished 2000 e e m u l 1600 o V eak ane P 1200 opent cycl kyl l A 800 + ee an ohex l c y c 400

kyl OMR011 - Thuleilat Shale l A OMR017 - Buah Shale

0

12 13 14 15 16 17 18 19 20 21 22 23 Total Carbon Number 3

2.5 OMR011 - Thuleilat Shale

ane) OMR017 - Buah Shale ent p o 2 cycl yl k l A // 1.5 ohexane cl yy c 1 kyl odddd c hihains >> even chhiains l A ( o ti aa R 0.5

0

13 14 15 16 17 18 19 20 21 22 23 Total Carbon Number Topics

• What are usefuluseful criteria forfor biogenicity? How can we be sure of measuring the right thing in a sample on Mars or returned from Mars? Deliberations of the 2000 NASA Biomarker Taskforce – molecular biosignatures • Analytical methods for investigating molecular biosignatures in rocks from Earth & elsewhere GC2-TOFMS • New ‘technologies’ in molecular biosignatures for organisms and ancient environments Hydropyrolysis & Strelllley Pool ChChert Hydropyrolysis (H2) Murchison thermocouple high pressure hydrogen reactor tube

electrical sample Hydropyrolysis connectors • sample impregnated - ammonium dioxydithiomolybdate - decomposes above 250 ºC ttoo ccatalystatalyst gas collection and measurement • continuous high flow of H2 • heated 220 to 520 ºC • ddryry ice ttraprap cold • dichloromethane trap pressure transducer Hydropyrolysis facilitates breakdown of macromolecules & minimises rearrangement δ13C profiles for HyPy products of 3 meteorites

5 Murch 0 -5 -10 -15 -20 -25 B)

DD -10 Orgueoil VP ,

ë -15 lues(¡

a -20 v CC 13

δ -25

Cold Bokk s -5 ne ee

diMeththy lbilbip henyl r y ne p

-10 yl h t e

-15 4H-naphthalene Acenaphthene Me ren methyl-biphenyl uoranthe l F Phenathrene/anthracene Py -20 800 1000 1200 1400 1600 1800 Retention time, sec Data: Mark Sephton, Cheng-Gong Sun, Gordon Love and Colin Snape δ13C n -Alkanes iinn MMeteoriteseteorites

-5 Orguiel (CI1) Vigarano (CV3) Cold Bokkeveld (CM2) Ornans (CO3) -10 Murchison (CM2) Bishunpur (LL3) -15 fraction action cc rr -20

-25

δ13 or aliphati C (‰) aliphatic f

PDB oo -30 hh

-35 ane-p ane-ric alk nes um alk n- aa

-40 -- ee n -45 Petrol Sephton et al., 2001 -50 Stahl 1979 leum n-alk

Bjoroy et al., 1991 oo Krishnamurthy et al., 1992 Petr

12 14 16 18 20 22 24 26 carbon number Microbes: Comparison of alkyl chain lengths (m/z 85)

18 16 Prochlorothrix hollandica 14 15 17 bacteria 1617 18 15 Phormidium luridum archaea algae 16 18 Chlorobium 17 20 thiosulfatophilum31 16 15 14 Methylococcus capsulatus 13 Ph Halobacterium saccharovorum Pr 16 17 Ph 16 18 Phormidium ‘RCO’ 15

18 Emiliania huxleyi 31 16 22 3738 14 33

16 18 Scenedesmus quadricauda 30 26 28 32

Retention Time Black Chert Breccia black chert veins and clasts

Complex ‘Dyke’ Breccia

SPC 16 Kerogen sequential HyPy

TICs 12 100

16 330 HyPy 14 15 13 % 18 17 Pr? Ph?

0 99% PAH & 70% alkanes in 14 Magnet EI+ 100 13 15 HT16 fraction 520 HPHyPy Phen 17 % 12 18 Py 19 20 12.44 0 Time 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00 32.50 35.00 37.50 40.00 42.50 45.00 47.50 50.00

Small EOP of n-alkanes in low T fraction probably indicates some younger contamination e.g. produced from reduction of even C no. fatty acids. Distribution of aromatic hydrocarbons from HyPy

C M 001 aro r e-sep 04 D ec 0 1 10 M a gnet EI + TIC 10 0 P 1 . 19 e5

Py McArthur River (HYC) MeP MePy MeN % more complex - more alkylated B(e) B(ghi) Py Per N Ch Co MeCo

0 Ti me 20. 00 30. 00 4 0.00 5 0.00 6 0.0 0 70. 00 80. 00 90. 00 100. 00

C M 003 A r o 04 D ec 0 1 05 M a gnet EI + TIC 10 0 Py 8 . 38 e4

Strelley Pool

% P MePy MeP b(e) B(ghi) Ch Py Per 9 Ti me 20. 00 30 .00 4 0.00 50. 00 60. 00 7 0.00 8 0.00 90. 00 m/z 178+ 192 m/z 202+ 216 P Py 100 MeP/P = 0.82 100

MeP C CM003 0.9 2 // R =0. 9257 % 2 % MePys 3 0.8 Fla 9 1 0.7 0 0 04 Nov 18 16 100 100100 o tomic H 0.6 ti aa

MeP /P = 0 .70 a CM002 0.5

% P r %

P/ 0.4 MeP/P and MePy/Py reflect bulke kerogen atomic H/C; M 0 000.3 reasing 04 Nov 18 15 cc 100 1001000.2

De CM004 MeP/P = 0 .35 0.1 Fla δ13 % %% C or [9+1]/[3+2] may ultimately0 help discriminate biology

0 Time 00 0 010.1 020.2 030.3 040.4 TiTi050mmee.5 24.00 25.00 26.00 27.00 28.00 29.00 30.00 31.00 32.00 33.00 34.00 35.00 36.00 330.0.0000 331.1.0000 332.2.0000 333.3.0000 334.4.0000 335.5.0000 336.6.0000 337.7.0000 338.8.0000 339.9.0000 440.0.0000 441.1.0000 442.2.0000 443.3.0000 444.4.0000 445.5.0000 H/C atomic ratio P = p henanthrene Fla = fluoranthene Me P = me thylphenanthrenes Py = pyr ene MePy = m ethyyplpyyre nes Concluding Thoughts 1. Organic compounds made by terrestrial organisms have generic structural & isotopic traits. Searching for these features in extraterrest rial OOMM i s a sound approach t o life d etecti on. 2. Terrestrial sediments as old as 2700Ma contain an abundance of ‘molecular biosignatures’ . Lipid biosynthetic pathways are of great antiquity & there is no evidence for there having been alternative pathways or extinct pathways. 3. Hydrocarbons are robust and, of all compound classes, are likely to be preserved under harsh conditions so long as they are sterile. Emerging technologies suchas mu ltidimensional GC and GC- TOF are useful analytical tools. 4. Hyropyrolysis assists screeningscreening forfor biosignatures inin macromolecular OM and biomass MIT OpenCourseWare http://ocw.mit.edu

12.458 Molecular Biogeochemistry Fall 2009

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