12.158 Lecture 6 • Steroids
– Structures and biosynthesis
– Diagenesis
– Steroidal hydrocarbons; stereochemistry vs
maturity
– Steroids as age and environment indicators
– Enigmatic steroids 2- and 3-alkyl and
carboxysteroids
1
Evolution of Hopane
p
& Sterol Bioynthesis
y
BHP
Squalene
p p
Diploptene
o2
O
BACTERIA
Squalene epoxide
EUCARYA
o2
HO
HO
Lanosterol
some bacteria - Methylococcus
Cholesterol
C24 substitution
by algae
Mycobacteria, Myxobacteria
2
Algal
g Steroids
•Encode a variety of age-diagnostic signatures –
C-isotopes + steroids from algae & plants
chlorophyceans
diatoms
H
HO
H
HO
chrysophytes
H
C29
C28
C30
HO
C30
dinoflagellates
H
HO
‘bio’
3
‘geo’
Functional Role of Sterols
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
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).
4
Do sterols & hopanoids serve the same membrane function?
HO
easy “flip“flip-flop
“fli
p-flop
flop”
OH
OH
unknown
unk
unkno
nown
wnpro
properties
per
ertie
ties
s
OH
OH
OH
OH
5
Fig. 4. Different proportions of cholesterol and CS in GUVs modulate domain size, domain curvatures, budding, and the formation of tubular structures
Bacia, Kirsten et al. (2005) Proc.
Proc Natl
Natl. Acad.
Acad Sci. USA 102,
102 3272
3272-3277
3277
Courtesy of National Academy of Sciences, U. S. A. Used with permission.
Source: Bacia, Kirsten et al. (2005) National Academy of Sciences,
6
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, Kirsten et al
Bacia
al. (2005) Proc
Proc. Natl
Natl. Acad.
Acad Sci
Sci. USA 102,
102 3272
3272-3277
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.�
7
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 "phytosterol" was proposed by Thoms in 1897 to
denote sterols of plant origin. The principal phytosterols are
compounds having 28 to 30 carbon atoms and include
campesterol, stigmasterol and β-sitosterol
http://www.cyberlipid.org/sterols/ster0003.htm
8
Sterols, Eukaryotes and O2
squale
squalen
ne epoxid
epoxide
e
squale
squalen
ne
1.14.
1.14.99.
99.7
7 + O2
22
21
5.4.
5.4.99.
99.7
7
5.4.
5.4.99.
99.8
8
12
CYCLO
CYCLOA
ARTENOLBR
BRANC
ANCH
11
19
1
2.1.
2.1.1.
1.41
41
3
1.14.
1.14.13.
13.70
70 + 3 O2
B
8
23
17
13
14
D
15
6
smt1
24--met
24
ethy
hyllene
necy
cycl
cloart
oarten
eno
ol
eburi
eburic
col
4,
4,4
4-di
dimet
meth
hyl-5?-chole
cholesta-8,
8,14,
14,2
24-tri
rien
en--3?-ol
HO
1.14.
1.14.13.
13.70
70 + 3 O2
smo1 + 3 O2 , 1.1.
1.1.1.
1.170,
170,1.
1.1.1.
1.1.270
270
HO
HO
1.3.
1.3.1.
1.70
70
4,4
,4--di
dimet
meth
hyl-5?-ergosta
ergosta8,
-8,
8,14,
14,24(28
14,2
28))-trie
trien
n-3?-ol
4,4
4,4-dimet
dimeth
hyl-5?-chole
cholesta-8,24
8,24--dien-3?-ol
HO
cy
cycl
cloeuc
oeucale
alen
nol
5.5.
5.
5.1.
1.9
9
HO
HO
1.14.
1.14.13.
13.72
72,,1.
1.1.
1.1.
1.170,
170, 1.
1.1.
1.1.
1.270
270 +3 O2
1.3.
1.3.1.
1.70
70
4,4-dimethylfecost
coste
erol
obtu
obtusifoli
folio
ol
1.14.
1.14.13.
13.70
70 + 3 O2
4?-methy
ethyllzy mosterol
mosterol
H O
HO
1.14.
1.14.13.
13.72
72,,1.
1.1.
1.1.
1.170,
170, 1.
1.1.
1.1.
1.270
270 +3 O2
HO
1.14.
1.14.13.
13.72
72,,1.
1.1.1.
1.1...170, 1.1.
1.1.1.
1.270
270 +3 O2
4?-met
ethy
hyllfec
eco
ostero
roll
zy
zymost
mosterol
erol
HO
1.14.
1.14.13.
13.72
72,,1.
1.1.1.
1.1...170, 1.1.
1.1.1.
1.270
270 +3 O2
4?-methy
ethyll-5?-ergost
rgosta
a-8,14,
14,2
24(28)
28)-tr
-trien-3?-ol
1.3.
1.3.1.
1.70
70
HO
HO
5.3.
5.3.3.
3.5
5
fecoster
stero
ol
4?-met
ethy
hyllfec
eco
ostero
roll
5.3.
5.3.3.
3.5
5
5?-chol
hole
esta-7,
7,24
24--dien-3?-ol
HO
HO
erg2
HO
1.3.
1.3.1.
1.72
72
24-methylenelophe
ophenol
nol
smt2
epi
epis
sterol
lathos
hostterol HO
HO
erg3+ O2
HO
1.3.3.2 + O2
5,
5,7,
7,24
24((28)-ergosta
rgostatrien
ieno
ol
7-dehydro
dehydro-chole
cholestero
eroll
HO
24--ethylene
24
nellophenol
smo2 + 3 O2 ,1.
1.1.
1.1.
1.170,
170, 1.
1.1.
1.1.
1.270
270
HO
HO
1.3.
1.3.1.
1.21
21
erg5 + O2
24
24--ethylen
enela
elatthostero
roll
5,
5,7,
7,22
22,24
,24((28)-ergos
gosta
tate
tetr
tra
aenol
chole
chol
estero
eroll
HO
1.
1.3.
3.3.
3.2
2 + O2
HO
HO
HO
1.3.
1.3.1.
1.71
71
ergostero
ergosteroll
24--met
24
ethy
hyllene55-dehyd
ehydroepis
roepisttero
roll
1.3.
1.3.1.
1.21
21
HO
HO
isofuc
ucost
oste
erol
dwf
dwf1
HO
12 O2
Burial & diagenesis
11 O2
11 O2
ergostane
cholestane
9
26
25
27
16
7
5
4
cloarten
eno
ol
cycloart
lanos
noste
terol
rol
10
A
HO
H O
9
2
PROTOSTEROLS
C
24
20
18
ANC
CH
LA
LANO
NOST
STERO
EROL
LBR
BRAN
29
28
O
si
sittost
ste
erol
HO
sitosterane
Sq
qualene Eppoxidase
O2
1 14 99 7
1.14.99.7
SQMO
squalene
squalene epoxide
O
O
N
HN
R N
FlredH2 + O2
O
O
N
O
O
NH O
FlH(4a)-OOH
N
R N
+
H
N
O
O
OH
NH
N
R N
Squalene
Floxreductase
+H
Regenerated by NADPH-c450
10
2O
O
H
O
N
O
+ (3S)-2,3-Oxidosqualene
Sq
qualene Eppoxidase
• SQMO Requires O2 (and FAD)
• K.
K Bloch and T
T.T.
T Tchen
Tchen, 1956 with
ith 18O2
• Electrophilic hydroperoxide reacts with 2,3-DB of squalene
• Water
Water is not a chemically feasible alternative to O2
• No evidence of any alternative oxygen-donors
• SQMO functionally conserved in Eukarya and Bacteria
O
O
N
HN
R N
FlredH2 + O2
O
N
O
O
NH O
FlH(4a)-OOH
N
R N
+
H
N
O
O
OH
NH
N
R N
Squalene Floxreductase
+H
Regenerated by NADPH-c450
11
2O
O
H
O
N
O
+ (3S)-2,3-Oxidosqualene
ENTRY EC 1.14.99.7
NAME squalene
l
monooxygenase
squalene epoxidase
squalene-2,3-epoxide cyclase
squalene 2,3-oxidocyclase squalene hydroxylase
squalene oxydocyclase squalene-2,3-epoxidase
poration of
CLASS Oxidoreductases Acting
gon paired donors with incorp
molecular oxygen Miscellaneous SYSNAME squalene,hydrogen
donor:oxygen oxidoreductase (2,3-epoxidizing) REACTION squalene +
2,3-epoxide
AH2 + O2 = (S)-squalene
(S) squalene-2
3 epoxide + A + H2O SUBSTRATE
SUBSTRATE squalene
AH2 O2 PRODUCT (S)-squalene-2,3-epoxide A H2O COMMENT A
flavoprotein (FAD). This enzyme, together with EC 5.4.99.7 lanosterol
synthase, was formerly known as squalene oxidocyclase
oxidocyclase.
synthase
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
squalene. J
J. Am
Am. Chem
Chem. Soc
Soc.
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.
12
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
sq
qualene 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 are necessary for enzyme activity
activity. 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
13
Oxidosq
qualene Cyyclase
O2
1 14 99 7
1.14.99.7
SQMO
squalene
O
squalene epoxide
5.4.99.7
OSC
5.4.99.8
PROTOSTEROLS
H
H
O
lanosterol
O
cycloartenol
14
ENTRY EC 5.4.99.7 NAME lanosterol synthase
2,3-epoxysqualene
lanosterol cyclase
squalene-2
squalene
-2,3
3-oxide-lanosterol
-oxide-lanosterol cyclase lanosterol
2,3-oxidosqualene cyclase squalene 2,3
epoxide:lanosterol cyclase 2,3-oxidosqualene
sterol cyclase oxidosqualene cyclase
2,3-oxidosqualene cyclase
2,3-oxidosqualene-lanosterol cyclase
oxidosqualene-lanosterol
id
l
l
l cyclase
l
squalene
l
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
15
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
16
Cytochrome
y
CYP51: Sterol 14α-demethylase
y
O2
O2
CH2OH
O2
CH(OH)2
O
HO
a
b
c
h
g
f
d
OH
HO
Fe+3
O
Fe+3
OH
O
O
Fe+3
e
-a cyctochrome P450 family enzyme
-ALL P450s have an absolute requirement for molecular O2
-CYP51
CYP51 requires
requires 3 molecules of O2 to activate and remove methyl
-the activated complex contains a porphyrin iron with an iron-oxo
species with formal oxidation state of Fe(V)!
17
O
O
Fe+3
The effect of oxygen on biochemical networks and the evolu:on of complex life. Jason Raymond and Daniel Segre' Science 311, 1764-­‐1767 (2006) Cholesterol This image has been removed due to copyright restric:ons. squalene hopene, tetrahymanol 18
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?
ergosterol
24-methyl-5,7,22-trien-3β-ol
HO
cycloartenol
HO
β-sitosterol
24-ethyl-5-en-3β-ol HO
HO
19
stigmasterol
24-ethyl-5,22-dien-3β-ol
Phytosterols
C30
y
y
HO
HO
HO ch
hollestt
HO
3β-oll 20
Microorganism
Major or common sterols
Microalgae
Mi
l
Bacillariophyceae
C28D5,22, C28D5,24(28), C27D5, C29D5, C27D5,22
Bangiophyceae
C27D5, C27D5,22, C28D7,22
C o op yceae
Chlorophyceae
C 8 5, C
C28D5,
C28D5,
8 5,7,,22,, C
C28D7,
8 ,22 C
C29D5,
9 5,22,, C
C29D5
9 5
Chrysophyceae
C29D5,22, C29D5, C28D5,22
Cryptophyceae
C28D5,22
Dinophyceae
4Me-D0, dinosterol, C27D5, C28D5,24(28)
E l
Euglenophyceae
h
C28D5 7 22 C29D5
C28D5,7,22,
C29D5, C28D7
C28D7, C29D5
C29D5,7,
7 C28D7
C28D7,22
22
Eustigmatophyceae
C27D5 (marine) or C29D5 (freshwater)
Haptophyceae
C28D5,22, C27D5, C29D5,22, C29D5
Pelagophyceae
g p y
, ( ), C29D5,,22,, C29D5,,C28D5 ,24((
28))
C30 D5,24(28),
Prasinophyceae
C28D5, C28D5,24(28), C28D5
Raphidophyceae
C29D5, C28D5,24(28)
Rhodophyceae
C27D5, C27D5,22
X th h
Xanthophyceae
C29D5 C27D5
C29D5,
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.
bonds In general,
general C28 sterols have a methyl group at C
C-24,
24 and C29
sterols have a 24-ethyl substituent. Table adapted from data in Volkman (1986); Jones et al.
(1994) and Volkman et al.
21
Uncommon Marine Sterols
HO
HO
24-methyl-27-nor sterols and stanol
known in sponges: C27 compounds
24-nor sterols
a range of algae & invertbrates: C26
R
H
HO
H
HOH2C
HH
A-nor sterols
sponges
19-nor sterols
R=H, CH3, C2H5, methylene
Δ22 trans unsaturation
probably formed by de-alkylation of algal sterols
22
Uncommon Marine Sterols (2)
HO
HO
Aplysterol
Gorgosterol
23
• 1: Lipids. 1995 Mar;30(3):203-19. Related Articles, Links
–
Developmental regulation of sterol biosynthesis in Zea mays
mays.
Guo DA, Venkatramesh M, Nes WD.
Department of Chemistry and Biochemistry,
Biochemistry Texas Tech University,
University Lubbock
79409, USA.
Sixty-one sterols and pentacyclic triterpenes have been isolated and characterized
b chromatographic
by
h
t
hi andd spectral
e t l methods
eth d from
f
Zea
Ze mays (corn).
(
) Several
Se e l plant
l t
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]mevalonic acid, [2-(14)C]acetate, and [2
(3)H] tatte)), andds tabl
(3)H]acet
t ble isottopes (D2O),
(D2O) we di
discovered
d that
th 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
h steroll sid
ide ch
haiin. Blad
Bl des synth
hesiized
d maiinlly 24a lph
l ha-ethhyl-sterol
l
ls, sh
heathhs
off the
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-ethy
yl-sterol indicated the Bloch-Cornforth route origginating
gwith
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.
24
Sterol ID by GC
GC-MS
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
acetate
Mass spectrum (m/z
(m/z, relative intensity to
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),
(18) 368 (6)
(6), 353 (13),
(13) 315 (16),
(16) 288 (7),
(7) 273 (6),
(6) 255 (21),
(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),
(12) 415 (2),
(2) 370 (33),
(33) 355 (16),
(16) 316 (3),
(3) 276
(28), 275 (18), 257 (4), 230 (17), 215 (100), 201 (18), 147 (32), 81
(53)
•
25
Sterols in Bacteria
Cyanobacteria
y
- many
y reports
p
Methanotrophs - Methylococcus capsulatus
Planctomycetes - Gemmata obscuriglobus
Myxobacteria - Nannocyctis exedans
• Likely false positives - cyanobacteria
• Unusual products - Gemmata
• Incomplete
p
pathways
p
y Methylococcus
y
• No alkylation at C24
• Potentially obtained from Eukarya by LGT
26
Sterol Biosynthesis in Bacteria ?
Molecular Microbiology (2003) 47(2), 471–481
Steroid biosynthesis in prokaryotes: identification of myxobacterial steroids and cloning
of the first bacterial 2,3(S)-oxidosqualene cyclase from the myxobacteriumStigmatella
aurantiaca Helge Björn Bode
aurantiaca.
Bode, Bernd Zeggel,
Zeggel Barbara Silakowski
Silakowski, Silke C
C. Wenzel
Wenzel, Hans
Reichenbach and Rolf Müller
Summary
Steroids,, such as cholesterol,, are sy
ynthesized in almost all eukary
yotic 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
intrig ing mechanisms involved
intriguing
in ol ed in triterpene biosynthesis
bios nthesis have
ha e attracted m
much
ch
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 biosy
ynthesis in bacteria. Only
y three steroid-sy
ynthesizing
g bacterial sp
pecies 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 the cloning of the first bacterial steroid biosynthesis gene,
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
comp
parison with wild typ
ype nor any
y increase in ethanol sensitivity.
y The p
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.
27
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
28
Phylogenetic and biochemical evidence for an ancient origin of stteroll bi
biosynthesi
th is in
i the
th Bactt
eriia
Ann Pearson,* Meytal Budin*, and Jochen J. Brocks† PNAS In Press
Press
• Abstract
Sterol biosynthesis is viewed primarily as a eukaryotic process, and the frequency
off it
its occurrence in
i bacteria
b t i long
l
h
has b
been a subject
bj t off controversy.
t
T
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
f eukaryotic
k
ti O
Osc were compared
d tto all
ll available
il bl complete
l t
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.
quent modifications of these products. In bacteria, the
There are no subseq
sterol biosynthesis genes occupy a contiguous coding region and possibly
comprise a single operon.
29
Biomarker Numbering System
21
12
14
1
22
17
10
C35 αβ-hopane
4
20
24
17
1
4
14
5
C29 ααα-sterane (20R)
30
Sterol Diagenesis
R
R
20
sterol
organisms
αα (20R)
sterene
immature
sediments
o17
o
o
14
o
HO
Backbone rearrangement and reduction
R
R
o
o
259
31
sterane
immature
sediments
Sterane maturity parameters
R
R
20
sterol
organisms
αα (20R)
o17
sterane
immature
sediments
o
o
o
14
HO
R
R
R
o
o
o
o
ββ (20S))
ββ (20R))
mature sediments & oil
32
αα (20S))
Steranes
mass spectral features
R2
= 372 +R+R2
217+
R
R
M+
259/257
262 + R2
257 17α(H) steranes
259 17β(H) steranes
218 > 217 in steranes with ββ config.
config
149/151 pair also sensitive to stereochemistry
33
Diasteranes
mass spectral features
R
M+
= 372 +R
259
34
Steranes - 217 Da
C29 Reg
C29Dia
Gippsland
C27Dia
NW Shelf
Middle East
35
Intternall Stand
St dard
ds ffor stterane quantit
titati
tion 217 Da•
D Æ 221 Da
D
36
80 60 60
0
48:00
50 40 40
50:00
R
C28 βα 20S, 24S +R
C27 βα 20R
70 52:00
30 α 20S
C28 ααα
+ Bicad T
54:00
37
TIME (min.)
56:00
20
58:00
C3
30 ααα 20R
C29 ααα 20R
C28 αββ 20R
R
C28
8 αββ 20S
+ Biicad T1
C28 α
ααα20R +
Bic
cad R
C29 αα
αα 20S
C29 α
αββ 20R
C29 αββ
β 20S
C27 ααα 20R
C29 βα 20R
C30
0 βα 20S
C27 αββ 20S
C
C29 βα 20S
+C27 αββ 20
0R
100 %
Bicad W
7 ααα 20S
C27
C2
28 βα 20R, 2
24S + R
90 7 βα 20S
C27
File A6MA15C:4
SIM-GCMS: 217.196 Da
Scale Factor: 4.9
AGSO Standard
217 Da
10 1:00:00
File A6MA15C:4
SIM-GCMS: 218.203
Da
Scale Factor: 4.1
90
80
60
50
C29 αβ
ββ 20S
9 αββ 20R
C29
C28 αββ 20
0R
70
C28
8 αββ 20S
100 %
C27
7 αββ 20S
C27 αββ
β 20R
AGSO Standard
218 Da
40 30 20 20
10 0
48:00 50:00
52:00
54:00
56:00
38
TIME (min.)
58:00
1:00:00
Steranes: SIM -GCMS vs. MRM -GCMS
C27 Dia
Total
C29 Dia+C27
C28
C27
C29
C30
C29
SIM :217 Da
C30
414 - 217
C29 400 - 217
C28 386 - 217 C27
372 - 217
39
40
C30 ααα 20R
2
C ααα 20R
C29
C29 αββ 20R
C29 αββ 20S
C29 ααα 20S
C28 ααα20R + Bicad R
C28 ααα 20S
+ Bicad T
C28
8 αββ 20R
C2
28 αββ 20S
+ Bicad T1
C30 βα
β 20S
C29 βα 20R
C ααα 20R
C27
Bicad W
C27 ααα 20S
0S
C29 βα 20
+C27 αββ
β 20R
C αββ 20S
C27
C28 βα 20R, 24S + R
C28 βα 20S, 24S +R
C βα 20R
C27
C27 βα 20S
AGSO Standard: steranes SIM vs. MRM
SIM-GCMS: 217 Da
MRM-GCMS: 400 -> 217 Da
MRM-GCMS: 386 -> 217 Da
MRM-GCMS: 372 -> 217 Da
21418 b/c +std 50ng/1000ul
03100216
100
%
61.32
57.57 58.62 59.91
56.11
%
57.12
0
03100216 2: MRM 24 Channels EI+
386.391 > 217.196
9.26e6
59.26 58.42
100
54.28
55 31
55.31
%
C29
58.76
55.39
58.19
0
03100216 61.22
2: MRM 24 Channels EI+
372.376 > 217.196
2.05e7
56 86
56.86
52.24
55.95
C27
53.10
%
C28
56.98
59.45
100
2: MRM 24 Channels EI+
400 407 > 217
400.407
217.196
196
1.87e7
61.20
61
20
100
C30
63.0064.23
55.95 56.86
52.24 53.10
0
03100216
2: MRM 24 Channels EI+
414.423 > 217.196 3.02e6
62.80 54.19
51.41
0
03100216
54.50 55.29
100 50.88
C26
54.30
%
0
52 26
52.26
52.50
55.97 56.86 58.25
55.00
2: MRM 24 Channels EI+
358.353 > 217.196
2.33e6
57.50
61.22
60.00
62.50
64.07
65.00
66.88
41
67.50
Time
70.00 MRM –GCMS
showinggsteranes
from a typical marine
sediment
L. Permian, Australia Note similarities in
isomer distributions
for each homolog
20S/20R
ααα/αββ
βα S+R/ααα+αββ
2- & 3-methylsteranes
21418 b/c +std 50ng/1000ul
03100216
62.11
100
60.82
%
414 423 > 231
414.423
231.212
212
62.82
63.06
58.6459.04
58.15
65 12 65.63
65.12
0
03100216
100
%
4-methylsteranes
62.80
414.423 > 217.196
61.32
57.57 58.62
59.81
55.95 56.86
52.24 53.10
MRM –GCMS
GCMS
showing C30 steranes of a marine sediment 63.00
64.23
0
03100216
100
61.20
400.407 > 217.196
61.14
404.432 > 221.221
59.91
56.11
%
57.12
58.76
55.39
0
03100216
100
%
0
51.57
52.50
55.02
55.00
58.23
57.50
64.05
60.00
62.50
65.00
42
67.50
Time
70.00
C26 steranes
t
21-nor
24-nor
27-nor
43
C26 steranes
el tion pattern
elution
pattern
This image has been removed due to copyright restrictions
restrictions.
Moldowan et al GCA 55, 1065, 1991
44
C30 Desmethylsteranes
h l
45
C30 Desmethyl Steranes Oil from Southern Oman
Oma
NZ Kora
24--n-propylcholestanes
24
414
217
OM20
24
24--i-propylcholestanes
414
217
59:00
1:00:00
1:01:00
46
ααα
20S
1:02:00
αββ
20R+S
ααα
20R
1:03:00
1:04:00
Time
C30 Desmethyl Steranes E t
Eastern
Sib
Siberia
i Oil
Oils
ES36
ES36
414
217
i/n>1
ES89
ES89
414
217
59:00
1:00:00
1:01:00
i/n<1
1:02:00
47
1:03:00
1:04:00
Time
48
Aromatic steroids
R
monoaromatic
ti desmethylsteroid
d
th l t
id
M+ --> 253 or m/z 253
H
R
maturity = TA/(MA+TA)
triaromatic desmethylsteroids
m/z 231
R3
R4
R1
triaromatic methylsteroids
m/z 245
R2
49
3
Aromatics
m/z = 245
245
3
Middle East
Mesozoic
Phosp
phoria
Permian
Ghadames
s
Frasnian
Volg
ga-Ural
Frasnian
50
3
3
3
triaromatic
dinosteranes
Distribution of
dinosteroids in
Phanerozoic
sediments
This imag
ge has been removed
due to copyright restrictions.
Moldowan and Talyzina
Science 281,168-1170, 1998
51
AGSOstd_vial2B 1 mg + 50 ng D4, 1/85 ul
03100229
61.34
61 24
61.24
100
2- & 3-methylsteranes
62.11
414.423 > 231.212
60.84
61.95
55.61
58.11
56.98
%
56 84
56.84
52.76
54.60
55.38
59.06
4-methylsteranes
62.85
59.22
60 41
60.41
57.40
63.06
56.56
63.20
65.14 65.38
0
03100216
62.11
100
60.82
61.95
414.423 > 231.212
4-methylsteranes
62.82
61.22
%
58 64
58.64
63.67
0
03100216
ααα-20S
100
57.57
%
52.24
0
50.00
52.00
55.95
53.10
54.00
56.00
56.86
414.423 > 217.196
61.32
60.15
62.13
60.80
57.99
58.00
65.12 65.38 66.05
62 80
62.80
61.81
61.95
59.81
63.99
αββ-20S+R
ββ
58.62
58.88
Why all the isomers?
63.06
60 54
60.54
59.87
58.15
56.96
59.04
60.00
62.00
ααα-20R
63.00
63.57 64.23
64.00
24-n-propylcholestane
52
65.12
66.00
68.00
Time
70.00
AGSOstd vial2B 1 mg + 50 ng D4, 1/85 ul
AGSOstd_vial2B
ααα-20S
αββ-20S+R
61.34
100
414.423 > 231.212
62.11
61.24
60.84
ααα-20R
61.95
58.11
59.06
% 56.98
58.65
57.40
57 52
57.52
59.22
59.68 59.95
58 80
58.80
57.67
61.58
60.94
61.81
62.85
60.41 60.55
60.11
63.06
62.45
63.20
63.74
dinosteroids
0
58.11
100
57.58
ααα-20S
58.63
59.83
%
58.51
58.90
59.46
57.12
61.42
61.16
59.68
414.423 > 217.196
61.81
62.81
61.32
60.15 60.39
59.20
αββ-20S+R
ααα-20R
61.95
62.11 62.25
60.76
63.06
63.16
24-n-propylcholestane
0
57.00
57.50
58.00
58.50
59.00
59.50
60.00
60.50
61.00
53
61.50
62.00
62.50
63.00
63.50
64.00
Time
2
2α-methyl-24-ethyl
th l 24 th l
OMR 026 SNA 1 mg+ 50 ng D4
αββ-20S+R ααα-20R
03073105
62.79
100
61.50
3β-methyl-24-ethyl
61.90
ααα-20S
%
59.68
59.44
414.423 > 231.212
62.63
61.22
63.50
63
50 63
63.76
76 60 63
60.63
65.87
0
03073105
62.79
100
62.27
61.06 61.88
60.95
60.57
61.20
%
56.59 56.79
63.68
63.48
63.99
65.87
57.50 58.2459.2559.40
59.98
0
03073105
65.00 61.06
100
414.423 > 217.196
60.95
66.13
61.88
60.57
%
400.407 > 217.196
59.42
56.4256.75
0
50.00
00
52 00
52.00
54.00
54
00
56.00
56
00
58.18 58.95
58 00
58.00
59.98
60 00
60.00
62 00
62.00
54
64.00
64
00
66 00
66.00
68.00
68
00
Time
70.00
70
00
2 & 3-alk
23 lkyllstteranes
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.
55
2- & 3-alkylsteranes
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 stigmastane (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.
56
2 & 33-alkylsteranes
2alkylsteranes
57
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
3β-alkyl
-alkyl steranes and 3-alkyl
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.
S t Netto, Má
Santos
Márciio R. M
Mell
llo and
d
Francisco De A.M. Reis
Org. Geochem., 26, 787-790
58
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 3βOH
Steroid incorporated in kerogen bound through C-3
as shown by chemolysis with deuterated reagents
59
Sterol Methyltransferases -SMT
These images have been removed
due to copyright restrictions.
- Responsible for alkylation at C24;
stereochemically precise with strict substrate
y
selectivity
-Use the 3β-OH group for recognition and
anchoring
60
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http://ocw.mit.edu
12.158 Molecular Biogeochemistry
Fall 2011
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61