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Home , Bangiophyceae, Thalassiosirales

12.158 Lecture 7

• Sterols part 2 – Sterol biosynthesis review and revision – Steroids as age and environment indicators – Enigmatic steroids 2- and 3-alkyl and carboxysteroids

1 squalenesqualene squaleneepoxide Sterols, and O2 29292929 1.14.99.7 + O2 28282828 OO

22222222 21212121 26262626 24242424 181818 20 23 25252525 1212 LANOSTEROL BRANCH 5.4.99.7 5.4.99.8 CYCLOARTENOL BRANCH 17171717 11111111 27272727 13131313 19191919 DDDD 16161616 1111 9999 CCCC 2222 14141414 PROTOSTEROLS 10101010 8888 15151515 AAAA BBBB 3333 7777 HOHO HOHO 5555 4 6 lanosterollanosterol cycloartenol

2.1.1.41 1.14.13.70+ 3 O2 smt1smt1

24-methylenecycloartenol

eburicoleburicol smo1 + 3 O2 , 1.1.1.170, 1.1.1.270

HOHO 4,4-dimethyl-5?-cholesta-8,14,24-trien-3?-ol HOHO HOHO

1.14.13.70+ 3 O2 1.3.1.701.3.1.70 cycloeucalenol

4,4-dimethyl-5?-ergosta-8,14,24(28)-trien-3?-ol 5.5.1.95.5.1.9 4,4-dimethyl-5?-cholesta-8,24-dien-3?-ol HOHO HOHO HOHO 1.14.13.72, 1.1.1.170, 1.1.1.270 +3 O 1.3.1.701.3.1.70 2 obtusifoliol

4,4-4,4dimethylfecosterol-dimethylfecosterol 4?-methyl zymosterol 1.14.13.70+ 3 O2

HOHO HOHO HOHO

1.14.13.72, 1.1.1.170, 1.1.1.270 +3 O2 1.14.13.72, 1.1.1..170, 1.1.1.270 +3 O2 4?-methyl-5?-ergosta-8,14,24(28)-trien-3?-ol

4?-methylfecosterol 1.3.1.701.3.1.70

zymosterolHOHO HOHO HOHO

1.14.13.72, 1.1.1..170, 1.1.1.270 +3 O2 5.3.3.55.3.3.5 4?-methylfecosterol 5.3.3.55.3.3.5 fecosterolfecosterol 5?-cholesta-7,24-dien-3?-ol HOHO HOHO HOHO erg2erg2 1.3.1.721.3.1.72 24-24methylenelophenol-methylenelophenol episterolepisterol smt2smt2 HOHO lathosterol HOHO HOHO

erg3+ O2 1.3.3.2 + O2 24-ethylenelophenol

5,7,24(28)--ergostatrienolergostatrienol smo2 + 3 O2 , 1.1.1.170, 1.1.1.270 HOHO 7-dehydro-cholesterol HOHO HOHO

erg5 + O2 1.3.1.211.3.1.21 24-ethylenelathosterol 5,7,22,24(28)--ergostatetraenolergostatetraenol 1.3.3.2 + O2 HOHO cholesterol HOHO HOHO 1.3.1.711.3.1.71 24-methylene 5-dehydroepisterol

ergosterolergosterol 1.3.1.211.3.1.21 HOHO

HOHO isofucosterol dwf1dwf1

HOHO 12 O 11 O 11 O sitosterolsitosterol 2 2 2 HOHO

Burial & diagenesis ergostane cholestane sitosterane

2 The effect of oxygen on biochemical networks and the evolution of complex life. Jason Raymond and Daniel Segre' Science 311, 1764-1767 (2006)

Cholesterol

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squalene hopene, tetrahymanol

3 http://prelude.bu.edu/ O2/networks.html

4 Oxidosqualene Cyclase

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5 Symbiogenesis: the phylogenetic tapestry of eukaryotes

http://www.life.umd.edu/ This image has been removed due labs/delwiche/ to copyright restrictions.

6 Diversity & Chloroplast Endosymbiosis

Anaerobes stem of tree?

Algae Forams Radiolaria

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©Jacob Waldbauer, 2007 Keeling et al. 2005 7 Phytosterols Whereas vertebrates and fungi synthesize sterols from epoxysqualene through the lanosterol, cyclize epoxysqualene to cycloartenol as the initial sterol. Q. Presumably lanosterol biosynthesis predates cycloartenol biosynthesis? What might have driven the lanosterol-cycloartenol bifurcation?

cycloartenol ergosterol HO β HO 24-methyl-5,7,22-trien-3 -ol

β-sitosterol stigmasterol β HO 24-ethyl-5-en-3β-ol HO 24-ethyl-5,22-dien-3 -ol

8 Phytosterols

C30 sterols are generally minor components of organisms and immature sediments. However, when they occur, they have distinctive structures that are easily recognised in the ancient record.

red algal sponge HO sterol HO sterol

4a-methyl-24-ethyl­ dinosterol HO HO cholestan-3β-ol

9 Oxidosqualene Cyclase Alignment

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10 Microorganism Major or common sterols Bacillariophyceae C28D5,22, C28D5,24(28), C27D5, C29D5, C27D5,22 Bangiophyceae C27D5, C27D5,22, C28D7,22 C28D5, C28D5,7,22, C28D7,22 C29D5,22, C29D5 Chrysophyceae C29D5,22, C29D5, C28D5,22 Cryptophyceae C28D5,22 Dinophyceae 4Me-D0, dinosterol, C27D5, C28D5,24(28) Euglenophyceae C28D5,7,22, C29D5, C28D7, C29D5,7, C28D7,22 Eustigmatophyceae C27D5 (marine) or C29D5 (freshwater) Haptophyceae C28D5,22, C27D5, C29D5,22, C29D5 Pelagophyceae C30 D5,24(28), C29D5,22, C29D5, C28D5,24(28) C28D5, C28D5,24(28), C28D5 Raphidophyceae C29D5, C28D5,24(28) Rhodophyceae C27D5, C27D5,22 Xanthophyceae C29D5, C27D5 : C27D5, C29D5, C27D0, C29D0 (evidence equivocal) Methylotrophic bacteria 4Me-D8 Other bacteria C27D5 Yeasts and fungi C28D5,7,22, C28D7, C28D7,24(28) Thraustochytrids C27D5, C29D5,22, C28D5,22, C29D5,7,22 The nomenclature is CxDy where x is the total number of carbon atoms and y indicates the positions of the double bonds. In general, C28 sterols have a methyl group at C-24, and C29 sterols have a 24-ethyl substituent. Table adapted from data in Volkman (1986); Jones et al. (1994) and Volkman et al.

11 Uncommon Marine Sterols

HO HO 24-methyl-27-nor sterols and stanol 24-nor sterols known in sponges: C27 compounds a range of algae & invertbrates: C26

R

H

HO HOH C H 2 H H 19-nor sterols A-nor sterols R=H, CH3,C2H5, methylene sponges Δ22 trans unsaturation probably formed by de-alkylation of algal sterols

12 Uncommon Marine Sterols (2)

HO HO

Aplysterol Gorgosterol

13 How to identify sterols by GC-MS of TMS and acetate derivatives

• Relative Retention Times of Nematode Sterols

• Mass Spectra of Nematode Sterols

• Mass Spectral Data for Nematode Sterols, Analyzed as Steryl Acetate Derivativesa Steryl acetate______Mass spectrum (m/z, relative intensity to base peak)__Cholesta-5,7,9(11)-trienol 424 (5), 364 (100), 349 (33), 251 (31), 209 (64), 197 (43), 195 (52)Cholest-8(14)-enol 428 (100), 413 (18), 368 (6), 353 (13), 315 (16), 288 (7), 273 (6), 255 (21), 229 (42), 213 (43), 81 (80), 55 (83)Cholesterol 368 (100), 353 (14), 260 (15), 255 (13), 247 (18), 213 (14), 147 (48), 145 (37), 81 (74), 55 (71) Cholestanol430 (12), 415 (2), 370 (33), 355 (16), 316 (3), 276 (28), 275 (18), 257 (4), 230 (17), 215 (100), 201 (18), 147 (32), 81 (53)

14 Sterol Reading #1

Text has been removed due to copyright restrictions.

Please see http://www.springerlink.com/content/q05172k241v60328

15 Reconstruction of past biota

16 Biomarkers and Fossils what can they can say:

Who are the major groups of marine primary producers today?

Which groups dominated at different periods in the geologic past?

How is growth recorded in rocks – This image has been removed and oil? due to copyright restrictions.

What factors influence the completeness of the fossil record of marine plankton?

How has long-term ecological succession of marine plankton affected the evolution of other organisms and biogeochemical cycles?

17 Marine Primary Producers

Today, 2 major groups:

Acquired photosynthesis by secondary endosymbiosis Were preceded by red/ & prokaryotic phototrophs These images have been Left a rich body & molecular fossil record removed due to copyright Rose to ecological prominence relatively restrictions. recently

Picocyanobacteria Chl a+c Prochlorococcus/ Marine Synechococcus Coccolithophorids

18 Eukaryote Diversity & Chloroplast Endosymbiosis

Anaerobes stem of tree?

Algae Forams Radiolaria

This image has been removed due to copyright restrictions.

©Jacob Waldbauer, 2007 Keeling et al. 2005 19 The Fossil Record of Phytoplankton - one form of bias: where sediment is deposited

20 Another form of Bias:Age of the Ocean Floor

200 0 NOAA NGDC

21 White Cliffs of Dover

Rocks Made of Plankton Cretaceous coccoliths

Diatomaceous Earth Mine, Wallace Co., Kansas K~T Boundary at Stevns Klint Denmark Grace Muilenburg, Kansas Geol. Surv. Courtesy of Organic Geochemistry Group, Universitat Bremen. Used with permission. 22 Succession in the Photosynthetic Plankton of the Ocean: A Perspective Drawn from Chemical Fossils

Roger E. Summons & Jacob R. Waldbauer (MIT) Andrew H. Knoll (Harvard University) John E. Zumberge (GeoMark Research) Successions in Biological Primary Productivity in the Oceans. In Falkwoski P. and Knoll A.H. (eds) The Evolution of Photosynthetic Organisms in the Oceans, 2006.

23 Evolutionary Trends from Rocks & Oils: Present-day sample localities

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24 Evolutionary Trends from Rocks & Oils • Sedimentary organic matter is the direct geologic legacy of primary production • Massive accumulations of organic matter in petroleum systems worldwide record ocean biogeochemistry • Oils are widely available, accessible, abundant & carry the same kind of evolutionary information that is buried in sediments • Oils reflect the natural ‘average’ in the variation in source rock organofacies • GeoMark Database: Biomarker parameters from over 1800 microbial-sourced oils (no terrigenous input) have been averaged to obtain 133 petroleum systems from the Neoproterozoic to Miocene • The source rock type and age for many of the oils in GeoMark’s database are known based on extensive integration of geological and geochemical frameworks

25 PaleoLatitude vs Carbonate-Sourced Oils 1.4 carbonate marl

1.2 shale

1.0

0.8 C29/C30H

0.6

0.4

0.2 -80 -60 -40 -20 0 20 40 60 80 Paleo Latitude

26 Pentacyclic Terpane Ratios 0.60

0.55

0.50

0.45

0.40

0.35 C31R/H 0.30

0.25 carbonate 0.20 marl shale 0.15 lacustrine

0.10 0.2 0.4 0.6 0.8 1.0 1.2 1.4 C29/H

27 • Clearly, these groups rise to paleontological prominence in Mesozoic and Cenozoic oceans, but… • Is there an earlier, “cryptic” evolutionary history? – Unmineralized or poorly mineralized stem diatoms or coccolithophorids? – Non-diagnostic fossils of dinos (sans archeopyle) among older acritarchs?? – Help from molecular clocks?? – Sedimentary record:Biomarker molecules are most informative

28 C28/C29 Sterane Ratios: The Rise of Modern Plankton

29 Biomarkers-Petroleum Record

1.0 carbonate marl 0.9 distal shale L12 DS21 lacustrine M1 C3 DS48 M4 DS47 DS54 M3 End Cretaceous 0.8 DS19 DS42 DS4 M2 L16 DS44 DS20 C5 DS5 C6 DS39 L13 End Permian DS16 DS2 DS9 L11 DS3 DS43 End Proterozoic DS13 L15 L20 DS53 C4 L14 0.7 DS10 DS15 DS40 DS45 DS1 DS34 L18 C15 C8 DS52 DS24 C2 DS12 DS37 L17 C14 DS46 DS8 DS26 DS38 C16 DS50 DS27 DS6 L19 DS51 C10 M9 DS65 C7 C9 C17 DS49 0.6 DS17 C1 DS41 C12 M6 DS14 M5 M8

DS11 C18 L24 0.5

L4 0.4

L7

Aromatic Dinosteranes (3/3+6) 0.3 L1 L6 L9 L3 0.2 L8 L2 L23 L5

0.1 DS56 L21 C21 DS57 DS68 DS70 M7 DS67 DS71 C19 C22 DS61 DS64 DS66 M10 DS72 C20 DS60 DS69 DS73 DS74 DS62 DS63 C25 0.0 DS55 DS59 0 50 100 150 200 250 300 350 400 450 500 550 600 Source Rock Age (mybp)

30 Aromatic dinosteroids/2+3-methyls due tocopyrightrestrictions. This imagehasbeenremoved 31 Science 281,168-1170, 1998 Moldowan and Talyzina dinosteroids in Distribution of Phanerozoic sediments C26 steranes

21-nor Unknown source

24-nor Unknown source

27-nor Unknown source

32 Secular Change in C26 Sterane Abundance Application of 24-norcholestanes for constraining source age of petroleum A. G. HOLBA et al. Org. Geochem. 29,1269 -1283, 1998 Rampen et al., AGU 2004:�����..Another specific biomarker is 24-norsterol. Its value as an age diagnostic biomarker was already reported (3), but the source of this sterol was still unknown although a diatomaceous source was assumed. We have now found this sterol in the species aff. Antarctica. In combination with the knowledge that the 24-norsterol production increased substantially during the Cretaceous this may provide a tool to predict the mutation rate of the Thalassiosirales. Our data show that molecular paleontology can assist in obtaining more reliable estimates of the molecular clock rate and thus be an important tool in reconstructing the evolution of diatoms.

24-nor HO 217 Diatom sterol Diatom sterane

33 C26 steranes elution pattern

This image has been removed due to copyright restrictions.

Moldowan et al GCA 55, 1065, 1991

34 Secular Change in C26 Sterane Abundance Application of 24-norcholestanes for constraining source age of petroleum A. G. HOLBA et al. Org. Geochem. Vol. 29, pp. 1269 -1283, 1998

Courtesy Elsevier, Inc., http://www.sciencedirect.com. Used with permission. 35 Diatom-specific HBI & their Geologic Occurrence Highly branched acyclic isoprenoid alkenes and alkanes

The Rise of the Rhizosolenid Diatoms

Jaap S. Sinninghe Damsté, Gerard Muyzer, Ben Abbas, Sebastiaan W. Rampen, Guillaume Massé, W. Guy Allard, Simon T. Belt, Jean-Michel Robert, Steven J. Rowland, J. Michael Moldowan, Silvana M. Barbanti, Frederick J. Fago, Peter Denisevich, Jeremy Dahl, Luiz A. F. Trindade and Stefan Schouten 23 APRIL 2004 VOL 304 SCIENCE

This image has been removed due to copyright restrictions. C20 HBI C25 HBI

•Identified in the diatom genera Rhizosolenia, Haslea, , and Pleurosigma only •Biological products have 1-6 unsaturations; fossils fully saturated

36 C30 Desmethylsteranes

37 C30 Desmethyl Steranes Oil from Southern Oman NZ Kora 24-n-propylcholestanes 414 217

αββ OM20 ααα 20R+S 24-i-propylcholestanes 20S ααα 414 217 20R

59:00 1:00:00 1:01:00 1:02:00 1:03:00 1:04:00 Time

38 C30 Desmethyl Steranes Eastern Siberia Oils

ES36 414 217 i / n > 1

ES89 414 217 i / n < 1

59:00 1:00:00 1:01:00 1:02:00 1:03:00 1:04:00 Time

39 Stratigraphic column of Huqf Supergroup withrepresentativ e lithology, biostratigraphy and geochronologica This image has been removed l constraints. due to copyright restrictions.

GD Love et al. Nature 457, 718-721 (2009) doi:10.1038/ nature07673

40 Catalytic hydropyrolysis (HyPy) biomarker geochemistry applications

� Pyrolysis assisted by high H2 pressure (15 MPa) and a molybdenum catalyst (active phase is MoS2)

� A powerful tool for releasing bound biomarkers

� high yields of biomarker hydrocarbons

� less structural/stereochemical alteration

� Love et al. (1995) Org. Geochem. 23, 981

� info on bonding (D2)

41 Hydropyrolysis apparatus

Thermocouple High pressure hydrogen Reactor tube Temp prog. amb. – 500oC @ 8oC/min

H 2 pressure Sample bed with 15 MPa Electrical dispersed Mo catalyst sweep gas flow connectors 10 dm3/min

Gas collection And measurement

Mass-flow controller Cold trap Pressure transducer

Temperature-programmed, open-system pyrolysis (fast residence time of volatile

42 Comparison of free and kerogen-bound hydrocarbons TICs of saturates for a Minassa-1(A1C) sample

05 May 17 10 17 100 19 std. * Free saturated hydrocarbons Ph 21 15 23 * = std.

25 % hopane/sterane region i18Pr 27 x x 29 x 31 x x x x x

2 05 May 31 08 100 17 19 HyPy of Kerogen 15 21 23 % Ph 25 hopane/sterane region i18 x 27 Pr x x x x 29 31 x x x x

2 Time 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00 80.00 85.00 90.00

Numbers refer to carbon chain lengths of n-alkanes X= series of mid-chain methylalkanes (unknown origin)

43 MRMGC-MS ion chromatograms

of C26–C30 desmethylsteranes released from catalytic hydropyrolysis of a Masirah Bay Formation (JF-1) and a Ghadir Manquil Formation (GM-1) kerogen. This image has been removed due to copyright restrictions.

GD Love et al. Nature 457, 718-721 (2009) doi:10.1038/ nature07673

44 45 21418 b/c +std 50ng/1000ul 2- & 3-methylsteranes 03100216 62.11 100 60.82 414.423 > 231.212 62.82 4-methylsteranes

% 58.6459.04 63.06 58.15 65.12 65.63

0 03100216 62.80 100 414.423 > 217.196 61.32 MRM –GCMS

58.62 % 57.57 showing C steranes 59.81 63.00 30 55.95 64.23 52.24 53.10 56.86 of a marine sediment 0 03100216 61.20 100 400.407 > 217.196 59.91 56.11 % 57.12 58.76 55.39 0 03100216 61.14 100 404.432 > 221.221

%

51.57 55.02 58.23 64.05 0 Time 52.50 55.00 57.50 60.00 62.50 65.00 67.50 70.00

46 2- & 3­ alkylsteranes

Note the exact

This image has been removed co-elution of the due to copyright restrictions. four synthetic isomers with the equivalent peaks of the Phosphoria oil

47 2- & 3­ alkylsteranes

Note the different isomer This image has been removed due to copyright restrictions. preference for marine vs lacustrine sediments

48 MIT OpenCourseWare http://ocw.mit.edu

12.158 Molecular Biogeochemistry Fall 2011

For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms.

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