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Home , Episterol, Isofucosterol, Parkeol, Sterane, Stigmastane

12.158 Lecture 4

• – Structures and – Diagenesis – Steroidal hydrocarbons; stereochemistry vs maturity – Steroids as age and environment indicators – Enigmatic steroids 2- and 3-alkyl and carboxysteroids Evolution of Hopane & Bioynthesis

BHP Dippploptene o2 BACTERIA

Squalene epoxide O

o2 EUCARYA

HO HO C24 substitution by algae some bacteria - Methylococcus Mycobacteria, Myxobacteria Algal Steroids •Encode a variety of age-diagnostic signatures – C-isotopes + steroids from algae & plants

H chlorophyceans HO C29

diatoms H HO C28

chrysophytes C30 H HO

dinoflagellates C30 H HO ‘bio’ ‘geo’ Functional Role of

These images have been removed due to copyright restrictions.

While it became clear very early that cholesterol plays an important role in controlling cell membrane permeability by reducing average fluidity, it appears now that it has a key role in the lateral organization of membranes and free volume distribution . These two parameters seem to be involved in controlling membrane protein activity and "raft" formation (review in Barenholz Y, Prog Lipid Res 2002, 41, 1). Do sterols & hopanoids serve the same membrane function?

HO

easy “fli p-fl op”

OH OH

unkno w npro pro ppe er tie s

O H OH Fig. 4. Different proportions of cholesterol and CS in GUVs modulate domain size, domain curvatures, budding, and the formation of tubular structures

Bacia , KKirstenirsten et al. (2005) PProcroc . NatlNatl. AAcadcad . Sci. UUSASA 102, 3272 -3277 Courtesy of National Academy of Sciences, U. S. A. Used with permission. Source: Bacia, Kirsten et al. (2005) National Academy of Sciences, USA 102, 3272-3277. Copyright (c) 2005, National Academy of Sciences, U.S.A.��

Copyright ©2005 by the National Academy of Sciences Fig. 2. The molecular structure of a sterol determines separation of phases in GUVs and the curvature of the lo phase

Bacia, KKirstenirsten et al. (2005) PProcroc . NatlNatl. AAcadcad. Sci. UUSASA 102 , 3272 -3277 Courtesy of National Academy of Sciences, U. S. A. Used with permission. Source: Bacia, Kirsten et al. (2005) National Academy of Sciences, USA 102, 3272-3277. Copyright (c) 2005, National Academy of Sciences, U.S.A.�

Copyright ©2005 by the National Academy of Sciences Trivia • Structural studies from 1900 to 1932, mainly by H.O. Wieland "on the constitution of the bile acids and related substances" (Nobel Prize Chemistry 1927) and by A.O.R. Windaus on "the constitution of sterols and their connection with the vitamins" (Nobel Prize Chemistry 1928), led to the exact structure and stereochemical configuration of cholesterol • The term "" was proposed by Thoms in 1897 to denote sterols of plant origin. The principal are compounds having 28 to 30 carbon atoms and include , and β-sitosterol http://www.cyberlipid.org/sterols/ster0003.htm squalene squaleneepoxide Sterols, and O2 29 1.14.99.7 ++ OO22 28 OO

22 21 26 24 18 20 23 25 12 LANOSTEROL BR ANCH 5.4.99.7 5.4.99.8 CYCLOCYCLOARTENOL BRANCH 17 BR BR 11 27 13 19 DDDD 16 1111 9999 CCCC 2222 14 PROTOSTEROLS 10 8888 15 AAAA BBBB 3333 7777 HOHO HO 5555 4444 6666 lanosterol ccycloartenol

2.1.1.41 1.14.13.70 ++ 33 OO22 smt1

2424--metethyhylleenecy cloartenol

eburicoll smo1 + 3 OO22 ,, 1.1.1.170,1. 1. 1.1.270

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

1.14.13.70 ++ 33 OO22 1.3.1.70 cycloeucalenol

4,4-- dimethyl--5?--ergosta--8,14,24(28)-- trien--3?--ol 5.5.1.9 4,4-dimethyl-5?-cholesta-8,24-dien-3?-ol HO HO HO 1.14.13.72, 1.1.1.170, 1.1.1.270 +3 O 1.3.1.70 1. 22 1.14.13.70 ++ 33 OO 4,4-dimethylfecosterol 4?-methylzymosterol 22 HOHO HO HO

1.14.13.72,1. 1. 1.1.170, 1.1.1.270 +3 O22 1.14.13.72,1. 1. 1.1..170, 1.1.1.270 +33 OO22 4?-methyl-5?-ergosta-8,14,24(28)-t-trien-3?-olol

4?-methylfecosterol 1.3.1.70 HO HOHO HO

1.14.13.72,1. 1. 1.1..170, 1.1.1.270 +3 O22 5.3.3.5 4?-methylfecosterol 5.3.3.5 5?-cholesta-7,24-dien-3?-ol HO HOHO HO erg2 1.3.1.72 2244--mmeetthhyylleenneellopheophenolnol epistterol smt2 HO HO HO

erg3+ OO22 13321..3..3..2 ++ O2 24-- ethylenelophenol

5,7,24(28)-ergostatrienol smo2 + 3 OO22 ,1. 1. 1.1.170, 1.1.1.270 HO 7-dehydro-cholesterol HO HO 1.3.1.21 erg5 + OO22 24-ethylenelathosterol 5,7,22,24(28)-ergostatetraenol 1.3.3.2 + OO22 HO cholesterol HO HO 1.3.1.71 2424--metethyhylleene5 5-dehydroepisisterorol

1.3.1.21 HO

HO dwf1

HO 12 O 11 O 11 O sitosterol 2 2 2 H O

Burial & diagenesis sitosterane Squalene Epoxidase

O2 111.1449.9997.7 SQMO O

squalene squalene epoxide

O O O H H N N N O HN O N O N O O O OH R N NH O R N NH R N N O

Fl H2 + O FlH(4a)-OOH Squalene O + (3S)-2,3-Oxidosqualene red 2 + 2

Regenerated by NADPH-c45Flox0 re +ducta H se Squalene Epoxidase

• SQMO Requires O2 (and FAD) 18 • K . BBlochloch and T. T. Tchen , 1956 with O 2 • Electrophilic hydroperoxide reacts with 2,3-DB of squalene

• WWaaterter is not a chemically feasible alternative to O2 • No evidence of any alternative oxygen-donors • SQMO functionally conserved in Eukarya and Bacteria

O O O H H N N N O HN O N O N O O O OH R N NH O R N NH R N N O

Fl H2 + O FlH(4a)-OOH Squalene O + (3S)-2,3-Oxidosqualene red 2 + 2

Regenerated by NADPH-c45Flox0 re +ducta H se ENTRY EC 1.14.99.7 NAME squalene monooxygenase squalene epoxidase squalene-2,3-epoxide cyclase squalene 2,3-oxidocyclase squalene hydroxylase squalene oxydocyclase squalene-2,3-epoxidase CLASS Oxidoreductases Actingon paired donors with incorporation of molecular oxygen Miscellaneous SYSNAME squalene,hydrogen- donor:oxygen oxidoreductase (2,3-epoxidizing) REACTION squalene + AH2 + O2 = (S)-squalene-232,3- epoxide + A + H2O SSUBSTRAUBSTRATE squalene AH2 O2 PRODUCT (S)-squalene-2,3-epoxide A H2O COMMENT A flavoprotein (FAD). This enzyme, together with EC 5.4.99.7 , was formerly known asas ssqualenequalene oxidocyclase . http://www.genome.ad.jp/dbget bin/www_bget?enzyme+1.14.99.7 Corey, E.J., Russey, W.E. and Ortiz de Montellano, P.R. 2,3-Oxidosqualene, an intermediate in the biological synthesis of sterols from squalene . J . AAmm . CChemhem . SSococ . 88 (1966) 4750-4751. 2 Tchen, T.T. and Bloch, K. On the conversion of squalene to lanosterol in vitro. J. Biol. Chem. 226 (1957) 921-930. 3 [UI:67015265] van Tamelen, E.E., Willett, J.D., Clayton, R.B. and Lord, K.E. Enzymic conversion of squalene 2,3­ oxide to lanosterol and cholesterol. J. Am. Chem. Soc. 88 (1966) 4752-4754. 4 [UI:70158491] Yamamoto, S. and Bloch, K. Studies on squalene epoxidase of rat liver. J. Biol. Chem. 245 (1970) 1670-1674. SUMMARY Rat liver microsomes previously heated to 50” for 5 mm accumulate 2,3-oxidosqualene on incubation with squalene. Squalene epoxidase activity can be assayed either with squalene and heated microsomes or with lO,ll-dihydro- squalene and intact microsomes. In common with other monooxygenases, the epoxidase requires TPNH and molecu­ lar oxygen. Both a soluble fraction of rat liver and micro- somes aarere necessary for enzyme aactivityctivity. Carbon monoxide or potassium cyanide fall to inhibit squalene epoxidation. The present paper reports some properties of t,he squalene epoxi- dase system and describes a heat treatment of rat liver micro- somes which abolishes cyclase activity without impairing the enzymatic conversion of squalene to 2,3-oxidosqualene.

Yamamoto, S. and Bloch, K. Studies on squalene epoxidase of rat liver. J. Biol. Chem. 245 (1970) 1670-1674 Oxidosqualene Cyclase

O2 111.1449.9997.7 SQMO O

squalene squalene epoxide

5.4.99.7 OSC 5.4.99.8

PROTOSTEROLS

H O H O lanosterol ENTRY EECC 5.4.99.7 NAME lanosterol synthase 2,3-epoxysqualene lanosterol cyclase squalene -232,3- oxide- lanosterol cyclase llanosterolanosterol 2,3-oxidosqualene cyclase squalene 2,3- epoxide:lanosterol cyclase 2,3-oxidosqualene sterol cyclase ooxidosqualenexidosqualene cyclase 2,3-oxidosqualene cyclase 2,3-oxidosqualene-lanosterol cyclase oxidosqualene-l anosterol cycl ase squalene epoxidase-cyclase CLASS Isomerases

Dean, P.D.G., Oritz de Montellano, P.R., Bloch, K. and Corey, E.J. A soluble 2,3-oxidosqualene sterol cyclase. J. Biol. Chem. 242 (1967) 3014­ 3015.

http://www.genome.ad.jp/dbget-bin/www_bget?enzyme+5.4.99.7 This image has been removed due to copyright restrictions. Please see the image on http://www.chem.qmul.ac.uk/iubmb/enzyme/reaction/terp/cholesterol.html

http://www.chem.qmul.ac.uk/iubmb/enzyme/reaction/terp/cholesterol.html Cytochrome CYP51: Sterol 14α-demethylase

O O O 2 CH2OH 2 CH(OH)2 2 O HO a b c d

OH OH O O h HO Fe+3 g O Fe+3 f O Fe+3 e O Fe+3

-a cyctochrome P450 family enzyme

-ALL P450s have an absolute requirement for molecular O2 -CYP51CYP51 requ ires 3 moleculesmolecules ofof O2 to activate andand remove methyl -the activated complex contains a porphyrin iron with an iron-oxo species with formal oxidation state of Fe(V)! The effect of oxygen on biochemical networks and the evoluon of complex life. Jason Raymond and Daniel Segre' Science 311, 1764-1767 (2006)

Cholesterol

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squalene hopene, tetrahymanol Phytosterols Whereas vertebrates and fungi synthesize sterols from epoxysqualene through the lanosterol, plants 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 Phytosterols

C30 y y

HO HO

HO HO cholest 3β-ol Microorganism Major or common sterols Mi croalgae Bacillariophyceae C28D5,22, C28D5,24(28), C27D5, C29D5, C27D5,22 Bangiophyceae C27D5, C27D5,22, C28D7,22 Chlorop hyceae C8C28D55,, C8C28D55,,7,22, C8C28D7,22 C9C29D55,,22, C9C29D5 Chrysophyceae C29D5,22, C29D5, C28D5,22 Cryptophyceae C28D5,22 Dinophyceae 4Me-D0, dinosterol, C27D5, C28D5,24(28) ElEuglenop hyceae C28D5 ,7 ,2222 , CC29D529D5 , CC28D728D7 , CC29D529D5 ,7 , C28D7 ,2222 Eustigmatophyceae C27D5 (marine) or C29D5 (freshwater) Haptophyceae C28D5,22, C27D5, C29D5,22, C29D5 Pelaggpyophyceae C30 D5,,()24(28),, C29D5 ,,,22, C29D5,C28D5 ,24(() 28) Prasinophyceae C28D5, C28D5,24(28), C28D5 Raphidophyceae C29D5, C28D5,24(28) Rhodophyceae C27D5, C27D5,22 XtXanthhhophyceae C29D5 , CC27D527D5 Cyanobacteria: 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 ssterolsterols 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. 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 Uncommon Marine Sterols (2)

HO HO

Aplysterol Gorgosterol • 1: Lipids. 1995 Mar;30(3):203-19. Related Articles, Links – Developmental regulation of ssterolterol biosynthesisbiosynthesis iinn Zea mays. Guo DA, Venkatramesh M, Nes WD. Department of Chemistry andand Biochemistry , TTexasexas Tech University, Lubbock 79409, USA. Sixty-one sterols and pentacyclic triterpenes have been isolated and characterized bhby chroma tograp hihic an d spe ctral methethods from ZeZea ma ys (corn ). SeSevera l plant parts were examined; seed, pollen, cultured hypocotyl cells, roots, coleoptiles (sheaths), and blades. By studying reaction pathways and mechanisms on plants fed radiotracers ([2-(14)C], [2-(14)C]acetate, and [2- (3)H]acetat e), ands t able isot opes (D2O), we discovered tha t hydroxymethylgutaryl CoA reductase is not "the" rate-limiting enzyme of sitosterol production. Additionally, we observed an ontogenetic shift and kinetic isotope effect in sterol biosynthesis that was associated with the C-24 alkylation ofhf the sterol sidide cha in. BlBlad es synthes ized mainlyl 2424a lp ha-ethylhl-stero ls, sheaths synthesized mainly 24-methyl-sterols, pollen possessed an interrupted sterol pathway, accumulating 24(28)-methylene-sterols, and germinating seeds were found to lack an active de novo pathway. Shoots, normally synthesizing (Z)- 24(28)-ethylidine-cholesterol, after incubation with deuterated water, synthesized the rearranged double-bond isomer, stigmasta-5,23-dien-3 beta-ol. Examination of the mass spectrum and 1H nuclear magnetic resonance spectrum of the deuterated 24-ethyl-sterol indicated the Bloch-Cornforth route originatingwith acetyl-CoA and passing through mevalonic acid to sterol was not operative at this stage of development. An alternate pathway giving rise to sterols is proposed. Sterol ID by GC- MS of TMS and acetate derivativesderivatives • 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 intensityintensity 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) Sterols in Bacteria

Cyanobacteria - manypy reports Methanotrophs - Methylococcus capsulatus - Gemmata obscuriglobus Myxobacteria - Nannocyctis exedans

• Likely false positives - cyanobacteria • Unusual products - Gemmata • Incomp lete p athway s Methy lococcus • No alkylation at C24 • Potentially obtained from EukaryaEukarya byby LGTLGT Sterol Biosynthesis in Bacteria ? Molecular Microbiology (2003) 47(2), 471–481 biosynthesis in prokaryotes: identification of myxobacterial steroids and cloning of the first bacterial 2,3(S)-oxidosqualene cyclase from the myxobacteriumStigmatella aurantiaca . Helge BjörnBjörn Bode , Bernd ZeggelZeggel , BBarbaraarbara Silakowski , Silke C . Wenzel, Hans Reichenbach and Rolf Müller Summary Steroids, such as cholesterol, are synthesized in almost all eukaryotic cells, which use these triterpenoid lipids to control the fluidity and flexibility of their cell membranes. Bacteria rarely synthesize such tetracyclic compounds but frequently replace them with a different class of triterpenoids, the pentacyclic hopanoids. The intrigu ing mechanismsmechanisms inv olv ed in triterpene biosy nthesis hav e attracted m uch attention, resulting in extensive studies of squalenehopene cyclase in bacteria and (S)­ 2,3-oxidosqualene cyclases in eukarya. Nevertheless, almost nothing is known about steroid biosynthesis in bacteria. Only three steroid-synthesizing bacterial species have been identified before this study. Here, we report on a variety of sterol-producing myxobacteria. Stigmatella Aurantiac is shown to produce cycloartenol, the well-known first cyclization product of steroid biosynthesis in plants and algae. Additionally, we describe thethe cloning of the firstfirst bacterial steroid biosynthesis gene, cas , encoding the cycloartenol synthase (Cas) of S. aurantiaca. Mutants of cas generated via sitedirected mutagenesis do not produce the compound. They show neither growth retardation in comparison with wild typype nor any increase in ethanol sensitivity. The protein encoded by cas is most similar to the Cas proteins from several plant species, indicating a close evolutionary relationship between myxobacterial and eukaryotic steroid biosynthesis. Sterol Biosynthesis in Bacteria Sterols of Methylococcus capsulatus

This image has been removed due to copyright restrictions.

Bird et al., 1971, Nature 230, 473-4 Phylogenetic and biochemical evidence for an ancient origin of sterol biosyn thes is in the Bact eria Ann Pearson,* Meytal Budin*, and Jochen J. Brocks† PNAS In Press • Abstract Sterol biosynthesis is viewed primarily as a eukaryotic process, and the frequency ofif itts occurrence in bacteria lon g has been a subbjjec t of controvers y. Two enzymes, squalene monooxygenase (Sqmo) and oxidosqualene cyclase (Osc), are the minimum necessary for initial biosynthesis of sterols from squalene. In this work, 19 protein gene sequences for eukaryotic Sqmo and 12 protein gene sequences for euk aryotti ic Osc were compared to allll ava ilil ablble comp lete and partial prokaryotic genomes. The only unequivocal matches for a sterol biosynthetic pathway were the a-proteobacterium, Methylococcus capsulatus, in which sterol biosynthesis already is known, and the planctomycete, Gemmata obscuriglobus. The latter species contains the most abbreviated sterol pathway yet identified in any organism. Experiments show that the major sterols in Gemmata are lanosterol and its uncommon isomer, parkeol. There are no subsequent modifications of these products. In bacteria, the sterol biosynthesis genes occupy a contiguous coding region and possibly comprise a single operon. Biomarker Numbering SSystemystem

21 12 17 22 14 1 10 4 αβ C35 -hopane

20 24 17

ααα 1 14 C29 -sterane (20R) 4 5 Sterol Diagenesis

R 20 R sterol o17 sterene organisms o o immature αα (20R) 14 o HO sediments

Backbone rearrangement and reduction R R

o sterane o immature 259 sediments Sterane maturity parameters

R R 20 sterol o17 organisms o sterane o o immature αα (20R) 14 HO sediments

R R R

o o o o

ββ (20S) ββ (20R) αα (20S) mature sediments & oil Steranes mass spectral features M+ R2

= 372 +R+R2 217+ R

259/257 R 262 + R2 257 17α(H) steranes 259 17β(H) steranes

218 > 217 in steranes with ββ config. 149/151 pair also sensitive to stereochemistry Diasteranes mass spectral features M+ R = 372 +R

259 Steranes - 217 Da

C Reg C29Dia 29 Gippsland

C27Dia NW Shelf

Middle East Internal StStan d ard s f or sterane quantitation 217 Da Æ• 221 Da File A6MA15C:4 SIM-GCMS: 217.196 Da AGSO Standard R

Scale FFactoractor: 4.9 00 2 217 Da 20S αββ βα 20S

100 % C29 +C27 d T α α

90 αα + Bica C28 20R 20S ββ 20S 80 βα αα β β 7 7 RR + αβ C2 20S 20R 20R C29

70 20S 20S R 20R βα C29 αββ 20R 20S ααα α 27 αββ ααα 20S, 24S cad T1 ααα 4S + βα 27 C27 W W ii αα

60 CC RR 8 8 7 7 22 C α βα 20 + B C2 C2 C29 C29 C29 Bicad 50 20R, C28 αββ 20S 20R + βα βα 8 ad R C28 αα 22 cc 0 0

40 αα C Bi C3 C28 30 20R ααα 0

20 33 C

10

0 48:00 50:00 52:00 54:00 56:00 58:00 1:00:00 TIME (min.) File A6MA15C:4 SIM-GCMS: 218.203 AGSO Standard Da 218 Da Scale Factor: 4.1 20R 20S 20R αββ β β β ββ 99 α αβ 100 % C2 20S C29 C27

90 αββ 7 7

80 C2 20S 70 αββ R 00 8 8 2

60 C2 αββ 50 C28

40

30

20

10

0 48:00 50:00 52:00 54:00 56:00 58:00 1:00:00 TIME (min.) Steranes: SIM -GCMS vs. MRM -GCMS

C29 Dia+C27 C27 Dia C29 C30 C27 C C Total 28 29 SIM :217 Da

C30 414 - 217

400 - 217 C29

C28 386 - 217

C27 372 - 217 AGSO Standard: steranes SIM vs. MRM SIM-GCMS: 217 Da 20R S R S 00 ββ 2 T 20 αβ +R 20R βα + R Bicad 20S ααα 20S 20R 20R 20S 20S αββ C29 +C27 20R 20S + Bicad 20S βα 20R 20R αββ C28 20R + ααα βα 20S, 24S αββ ααα C29 ααα βα αββ 20S 20R, 24S ααα C27 7 27 9 27 αββ 8 Bicad W α C29 βα 27 ααα 0R 29 9 α Bicad T1 22 22 2 2 CC C C β β 88 22 β CC CC C C C + C2 C2 C28 C28 C30 C28 ααα C30

MRM-GCMS: 400 -> 217 Da

MRM-GCMS: 386 -> 217 Da

MRM-GCMS: 372 -> 217 Da 21418 b/c +std 50ng/1000ul 03100216 2: MRM 24 Channels EI+ 62.80 100 414.423 > 217.196 61.32 3.02e6

58.62 % 57.57 55.95 63.0064.23 C 52.24 53.10 56.86 30 MRM –GCMS 0 03100216 2: MRM 24 Channels EI+ showing 61 .2200 100 440000 .407 > 217 .196 steranes 1.87e7 59.91 56.11 from a typical marine % 57.12 C 58.76 29 55.39 sediment 0 03100216 2: MRM 24 Channels EI+ 59.26 L. Permian, Australia 100 386.391 > 217.196 58.42 9.26e6 54.28 58.19 55 .31 % 56.98 C 28 59.45 61.22 Note similarities in 0 03100216 2: MRM 24 Channels EI+ isomer distributions 56.86 100 372.376 > 217.196 52.24 2.05e7 55.95 for each homolog 53.10 % C27 54.19 20S/20R 51.41 0 03100216 2: MRM 24 Channels EI+ ααα/αββ 55.29 358.353 > 217.196 100 50.88 54.50 2.33e6 βα /ααα+αββ 54.30 C26 S+R % 52 .2266 55.97 56.86 58.25 61.22 64.07 66.88

0 Time 52.50 55.00 57.50 60.00 62.50 65.00 67.50 70.00 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 marinemarine 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 C26 steranes

21-nor

24-nor

27-nor C26 steranes elu tion patternpattern

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Moldowan et al GCA 55, 1065, 1991 C30 Desmethlhylsteranes C30 Desmethyl Steranes Oil from Southern OmanOma NZ Kora 24--- n ---propylcholestanes 414 217

αββ OM20 ααα 20R+S 2424--ii--propylcholestanespropylcholestanes 20S20S ααα 414 217 20R20R

59:00 1:00:00 1:01:00 1:02:00 1:03:00 1:04:00 Time C30 Desmethyl Steranes EtEastern SSibiberi a OOilils

ES36 414 217 ii/ / 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

Aromatic steroids

R monoaromatic desme ththy ls teroidid M+ --> 253 or m/z 253

H maturity = TA/(MA+TA) R

triaromatic desmethylsteroids m/z 231

R3

R4 triaromatic methylsteroids m/z 245 R1 R2 3 Aromatics triaromatic m/z = 245 3 3 dinosteranes 3 3 Middle EastEast Mesozoic

Phosphoria Permian

Ghadames Frasnian

Volga-Ural Frasnian Distribution of dinosteroids in This image has been removed Phanerozoic due to copyright restrictions. sediments

Moldowan and Talyzina Science 281,168-1170, 1998 AGSOstd_vial2B 1 mg + 50 ng D4, 1/85 ul 03100229 61.34 2- & 3-methylsteranes 100 62.11 61 .24 414.423 > 231.212 60.84 61.95

55.61 58.11 4-methylsteranes % 56.98 59.06 62.85 59.22 56 .84 60 .41 57.40 63.06 52.76 56.56 63.20 54.60 55.38 65.14 65.38

0 03100216 62.11 100 60.82 61.95 414.423 > 231.212 62.82 4-methylsteranes 61.22

% 59.04 Why all the isomers? 58 .64 60 .54 63.06 59.87 56.96 58.15 63.67 63.99 65.12 65.38 66.05

0 03100216 αββ -20S+R62 .80 100 ααα -20S 61.32 414.423 > 217.196

61.81 61.95 % 57.57 58.62 ααα-20R 60.15 59.81 58.88 62.13 63.00 56.86 63.57 64.23 52.24 55.95 57.99 60.80 53.10 65.12

0 Time 50.00 52.00 54.00 56.00 58.00 60.00 62.00 64.00 66.00 68.00 70.00 24-n-propylcholestane AGSOstd_ vial2B 1 mg + 50 ng D4, 1/85 ul αββ-20S+R 61.34 100 ααα-20S 62.11 61.24 414.423 > 231.212 60.84

61.95 ααα-20R 61.81 58.11 61.58 59.06 % 56.98 60.94 62.85 58.65 59.22 59.68 60.55 57 .52 58 .80 59.95 60.41 57.40 57.67 60.11 63.06 62.45 63.20 63.74 dinosteroids 0

58.11 100 57.58 αββ-20S+R 58.63 ααα 414.423 > 217.196 -20S 61.81

62.81 59.83 61.32 ααα % 61.42 -20R 58.51 60.15 60.39 61.95 58.90 59.20 59.46 61.16 57.12 59.68 62.11 60.76 62.25 63.06 63.16 24-n-propylcholestane 0 Time 57.00 57.50 58.00 58.50 59.00 59.50 60.00 60.50 61.00 61.50 62.00 62.50 63.00 63.50 64.00 2 α-meth yl-24-e thyl OMR 026 SNA 1 mg+ 50 ng D4 αββ 03073105 -20S+Rααα 100 62.79 -20R

61.50 61.90 3β-methyl-24-ethyl % ααα-20S 62.63 414.423 > 231.212 61.22 59.68 63 .50 59.44 60 .63 63 .76 65.87 0 03073105 100 62.79

62.27 63.68

61.06 61.88 63.48 % 60.95 60.57 414.423 > 217.196 61.20 63.99

57.50 59.2559.40 65.87 56.79 58.24 66.13 56.59 59.98 65.00 0 03073105 61.06 100 61.88 60.95 60.57

% 400.407 > 217.196 59.42

56.42 59.98 56.75 58.18 58.95 0 Time 50 .00 52 .00 54 .00 56 .00 58 .00 60 .00 62 .00 64 .00 66 .00 68 .00 70 .00 2- &3& 3-alkylsteranes

Text has been removed due to copyright restrictions. Please see: Abstract, JoséA. D. Lopes et al. "Geosteranes: Identification and Synthesis of a Novel Series of 3-Substituted Steranes." Organic Geochemistry 26, no. 11-12 (July 1997): 787-790. 2- &3& 3-alkylsteranes

INTRODUCTION

Since the first reports of 3-methyl (Summons and Capon, 1988; Summons et al. 1988), and 3-carboxyl- steranes (Dany et al., 1990), other sterane bio-markers substituted at the C-3 positions have been reported (Summons and Capon, 1991; Dahl et al., 1992; Lopes et al., 1992, 1994; Schaeffer et al., 1993, 1994) belonging to the 5α(H) series of cholestane (R2 = H), ergostane (R2 = Me) and (R2 = Et). These compounds are of geological interest because they have unusual substitution patterns and because logical precursors have not been identified in any living orghanism. 2- &&3 3 -alkylsteranes

Courtesy Elsevier, Inc., http://www.sciencedirect.com. Used with permission. Fossil steranes no longer have the original oxygen at C3

Fossil steranes with unprecedented methylation in ring-A, 1988. Roger E. Summons and Robert J. Capon Geochimica et Cosmochimica Acta 52, 2733-2736

Identification and significance of 3β-ethyl steranes in sediments and petroleum, 1991. Roger E. Summons and Robert J. Capon GCA, 55, 2391-2395

Extended 3β -alkyl steranes and 3 -alkyl triaromatic steroids in crude oils and rock extracts 1995. Jeremy Dahl, J. Michael Moldowan, Roger E. Summons, Mark A. McCaffrey, Paul Lipton, D. S. Watt and Janet M. Hope GCA, 59,, 3717-3729

Geosteranes: Identification and synthesis of a novel series of 3­ substituted steranes. 1997. JoséA. D. Lopes, Eugênio V. StSantos Neto, MMáárci o R. M ellllo an d Francisco De A.M. Reis Org. Geochem., 26, 787-790

Courtesy Elsevier, Inc., http://www.sciencedirect.com. Used with permission. Diagenetic Pathways of Sterols

Δ2-sterenes are primary sterol dehydration products as shown in lab expts. and data from immature sediments. Nature 269, 978, 1977 Diverse 3-alkylated steranes direct evidence of 3β- OH

Steroid incorporated in kerogen bound through C-3 as shown by chemolysis with deuterated reagents Sterol Methyltransferases -SMT

These images have been removed due to copyright restrictions.

- Responsible for alkylation at C24; stereochemically precise with strict substrate selectivity -Use the 3β-OH group for recognition and anchoring MIT OpenCourseWare http://ocw.mit.edu

12.458 Molecular Biogeochemistry Fall 2010

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