12.158 Lecture 7

• More on cyclic terpenoids – Analytical methods for hopanoids – LCMS Insights – Tricyclic and tetracyclic terpanes – Plant sesqui-, di-, triterpenoids

11 known ring systems

BHP Ring Configurations C-2 Me CYANOBACTERIA

Summons et al., 2000

C-3 Me METHANOTROPHS (Acetic Acid bacteria)

2

3

22R

Δ6

Δ11

Δ6 and/or Δ11

ACETIC ACID BACTERIA (Methanotroph)

HelenTalbot_nrg

3031

BHP Side-Chain Structures Composite Z OH OH NH2

O OHY OH X

HO OH OH

NH2H NH2N

TETRA: X=OH, NH2, composite; Y = Z = H O OH

PENTA: X = OH, NH2, composite; Y = OH, Z = H X = OH, Y = H, Z = OH COOH

HEXA: X = NH2; Y = Z = OH

O

O

OH

OH

HelenTalbot_nrg

Analysis of BHP

• Highly functionalised, amphiphillic • Not amenable to conventional GC-MS • Side chain cleavage (Rohmer et al., 1984)

• Periodic acid/sodium borohydride • Product structure directly related to number

and position of functional groups in side chain

• Specific nature of functional groups lost

HelenTalbot_nrg

HelenTalbot_nrg

OH

OH OH

OHOH 31

OH

OH

OH OH

NH2OH

OH 30

OH

O O

H

OH

O

OHOOH OH

OH

32 OH

TETRA

PENTA

32-hopanol H5IO6/NaBH4

HEXA

Rohmer periodic acid oxid & red

31-hopanol H5IO6/NaBH4

30-hopanol H5IO6/NaBH4

Talbot LCMS Methodology � Extraction:

• ultrasonication & Soxtherm in 2:1 chloroform/ MeOH

� Acetylation (acetic anhydride/pyridine) � RP-HPLC (Adapted from Schullenberg-Schell et al.,

1989)

•15 cm C18 column with 1 cm guard column •Ternary solvent system

Time (min) 0-5 10 40 70 75 Water (%) 10 5 1 1 10 Methanol (%) 90 80 59 59 90 Propan-2-ol (%) 0 15 40 40 0

� Positive ion APCI (Talbot et al., 2001 J. Chromatogr. A)

HelenTalbot_nrg

A/B Fragment R = H 191 R = CH3 205

D/E R3=H 191

A/B Fragment R = H 191 R = CH3 205

D/E 277

Mehay Modifications to Talbot et al APCI LC-MS

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Elucidating Hopanoid Biosynthesis with Mutant Bacteria

Proposed hopanoid biosynthetic pathway

shchpnC hpnE hpnG 4258 4257 hpnH ispH hpnN14266hpnN2hpnPeutB hpnO

eutC 4265 hpnD 4259 1 kb

IspH HpnP

HpnE?

Shc

HpnG

HpnH

HpnO

HpnP

HpnP

Biosynthesis Not biosynthetic

Not verified Transport

15 16 17 18 19 20 21

TIE-­‐1  diploptene  

2-­‐methyldiploptene  

15 16 17 18 19 20 21

Δ hpnP   diploptene  

15 16 17 18 19 20 21

Time (minutes)

Δshc    squalene  

Triterpenes  in  shc  and  ΔhpnP  mutants  

Deletion of HpnH Only C30 (core triterpenoids) present in this mutant

Deletion of HpnG Only adenosylhopane present in this mutant

Normal complement of BHP present in this mutant

accumulation of ribonyl hopane in the mutant. Formation of aminotriol

appears blocked.

Novel unknown BHP in addition to the normal

complement of BHP present in this mutant

James Saenz PhD: Panama Liquid Jungle Transect

James Saenz PhD: Panama Liquid Jungle Transect

Other sedimentary hopanoids

Hopanoic acids in Mesozoic sedimentary rocks: their origin and relationship with hopanes Paul Farrimond, Tony Griffiths, Efthymios Evdokiadis Organic Geochemistry 33 (2002) 965–977

Fig. 4. An m/z 191 mass chromatogram (top) showing the distribution of hopanoic acids (as methyl esters) in a Triassic sample (Serpiano shale) from Switzerland. The m/z 470, 484, 498, 512 and 526 mass chromatograms represent the molecular ions of C 3135 hopanoic acid methyl esters, including A-ring methylated hopanoic acids (shaded peaks; m/ z 205) which are labelled ��Me�� and with the numerical code oftheir nonmethylated equivalent hopanoic acid

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

Bisnorhopanes �Dinorhopanes OMO 002 SNA 1mg+ 50 ng D4, 1/100 ul 03072223 2: MRM 26 Channels EI+ 100 66.14 426.421 > 191.179

66.40 7.53e8

%

66.8967.1367.9868.4064.100 03072223 2: MRM 26 Channels EI+

63.98 412.406 > 191.179100 1.03e928,30-dinorhopane %

64.14 66.65 64.4264.77 66.14

0 03072223 2: MRM 26 Channels EI+ 100 62.20 398.39 > 191.17929,30-dinorhopane 1.03e9

% 62.30 62.9363.17 64.00

0 03072223 2: MRM 26 Channels EI+ 100 61.17 62.18

2.36e8 59.84 384.374 > 191.179

% 62.30

55.5355.87 58.54 59.51 60.42 61.67 63.17 64.000

03072223 2: MRM 26 Channels EI+ 100 59.17 370.359 > 191.179

7.45e8

% 58.30

59.920 Time 55.00 56.00 57.00 58.00 59.00 60.00 61.00 62.00 63.00 64.00 65.00 66.00 67.00 68.00 69.00 70.00

Bisnorhopanes �Dinorhopanes

28,30-dinorhopane: free hydrocarbon in highly anoxic sediments; Monterey Fm., Kimmeridge F.m, U-Shale

29,30-dinorhopane; member of homologous series of 3­norhopanes; direct biological precursors? High in carbonates

H

30-nor-αβ-hopane 384.374 > 191.179

Birba 3 (sapropelic laminite) Bisnorhopanes �Dinorhopanes Stalvies, Love

29αβ�

a) Kerogen-bound hopanes Tm

m/z 191

60.00 62.00 64.00 66.00 68.00 70.00 72.00 74.00 76.00 78.00 80.00 82.00

30αβ�

S RS

S S

S RR

RR

gammacerane

30αβ�29

αβ�

b) Free hopanes

60.00 62.00 64.00 66.00 68.00 70.00 72.00 74.00 76.00 78.00 80.00 82.00

gammacerane

28D

N

Tm

S R

S R

S R

S R

S R

Ts NB C29H>C30H

m/z 191

Other Dinorhopanes and Trinorhopane

H 25, 28, 30-trinor-αβ-hopane oftenfoundwith28,30-dinorhopane sometimes referredtoasC27T major ions 370M+, 177A+B, 163D+E

25, 30-dinor-αβ-hopane biodegradationproduct of 30-nor-αβ-hopane major ions 384M+, 177A+B, 163D+E

Other Triterpenoids- Rearranged Hopanoids AGSOstd_vial2B 1 mg + 50 ng D4, 1/85 ul03100229 2: MRM 24 Channels EI+ 100 65.12 426.421 > 191.179

6.03e765.38

%

63.04 63.9264.09 65.8466.0766.870

03100229 2: MRM 24 Channels EI+63.04 412.406 > 191.179100 17α(Η)� 1.34e8

% diahopane 60.13 60.84 61.72

62.81 63.2063.8264.7765.120

03100229 2: MRM 24 Channels EI+61.24 398.39 > 191.179100 18α(Η) -22,29,30-trisnor 18α(Η)-30-nor 1.53e8

% neohopane (Ts) neohopane (C29Ts) 61.36 55.63 59.79 62.23 63.04

003100229 2: MRM 24 Channels EI+ 100 61.24 384.374 > 191.179

2.05e758.92

% 59.12 61.36 55.9156.54 57.6158.1558.61 59.7760.2360.86 62.23 63.04

003100229 2: MRM 24 Channels EI+ 100 57.38 58.25 370.359 > 191.179

6.20e7 58.11 %

55.6356.1356.54 58.53 59.20

0 Time 55.00 56.00 57.00 58.00 59.00 60.00 61.00 62.00 63.00 64.00 65.00 66.00 67.00 68.00 69.00 70.00

Rearranged Hopanes

αβ(22S) hopane

some mature sediments

& oil

17α(Η)� diahopane

18α(Η) -22,29,30-trisnor neohopane (Ts)

18α(Η)-30-nor neohopane (C29Ts)

mature sediments & oil

H H

0.30

0.40

Rearranged Hopanes Source and Maturity Indicators C

29 T

s/T

m

0.20

0.10

0.00

SOUTHERN MARGIN OIL FAMILIES

0.0 0.2 0.4 0.6 0.8 1.0 C27 Ts/Tm

Kangaroo Is. StrandOB OB SawpitBass GA1 GA2 GB Migr

C35

S/C

34S

Hop

ane

1.6

1.4

1.2

1.0

2.2

2.0

1.8

0.8

0.6

0.4

0.2

Asphaltite

Marine Shale Paralic Shale Carbonate Marl EvaporiteCoal/ResinLacustrine

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

C29/C30 Hopane

Tricyclic Terpanes

Tricyclics or cheilanthanes extend to C40 or C45mostly marine and mature

regular isoprenoid branching (not like squalene) unknown source organism

Tricyclic Terpanes

m/z 191

Tricyclic Terpanes

UT031

UT043

Extended Tricyclic Terpanes in Petroleum

AL007 Silurian Marine

20min 95.45min

AN160 Early Cretaceous Lacustrine

C20

C21

C22

C23

C24

C25 *

C30 **

C35 ***

C40? ****

C26

C27?

C28 C29

Ts Ts

diahopane C38

C32

C37? C41RT �jump�

C31

C33

C34

C36 C39

C42

C31H 20R

* = # asymmetric carbons in side chain

C19

/C23

Tric

yclic

Ter

pane

s 19

/23

(Tri

cycl

ic T

erpa

nes)

Tricyclic Terpanes Are Powerful Lithology

Indicators in Sediments & Oils 10.00

terrestrial

1.00

0.10

marine

0.01 0.00 2.00 4.00 6.00 8.00 10.00 12.00

Pr/Ph (Pristane/Phytane)Pristane/Phytane

0.2

0.4

0.6

0.8

1.0

1.2

1.4 T

ricy

clic

Ter

pane

C24

/C23

Marine Shale

Tricyclic Terpanes Are Powerful Lithology Indicators in Sediments & Oils

Paralic Shale Carbonate Marl Evaporite Coal/Resin Lacustrine

Asphaltite

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

Tricyclic Terpane C22/C21

0.7

Diagnostic Tricyclic Terpane Ratios 1.3

Marine Distal Shales 1.1 Paralic/Deltaic Shales

Carbonates Marls0.9

TT

C24

/C23

Coals/ResinsLacustrine

0.5

0.3

0.1 0.0 0.5 1.0 1.5 2.0 2.5

TT C22/C21

Application to Oman Oils – Nafun (Pre-salt) versus Ara (Intra- salt) biomarker parameters

C22

T/C

21T

1.00

Athel domain0.80

Exceptions 0.60

0.40

Carbonate Stringers

0.20

Pre-salt domain

0.00

A1C

A2C

A3C

A4C

A5C

Buah

Masirah Bay

Shuram

Silicilyte

Thuleilat

U Shale

0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80

C24T/C23T

Diagnostic Hopane vs Tricyclic Terpane Ratios

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

C31

R/C

30 H

opan

e

Marine Shale

Carbonate

Marl

Lacustrine

Asphaltite

0.5 0.7 0.9 1.1 1.3 1.5 1.7 1.9

Tricyclic Terpane C26/C25 2.1

C24 Tetracyclic Terpane

191 Da mass chromatogram (above) and partial m/z 191 mass chromatogram (below). Peaks in the partial mass chromatogram are normalized to the C24 tetracyclic terpane -- the most abundant terpane in the data. The oil shown is a lacustrine oil from the Cuyo Basin, Argentina.

Principal Component Y 19/23 Pr/Ph

analysis of 800 oils scores & loadings

for OilMod variables 31R/H 27/17

29/H22/21

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Z 26/25

OL/H

Marine Shales

Paralic/Deltaic Coal/Resinitic

LacustrineCarbonate/ Marl

Hypersaline

X

24/23

35/34

S/H GA/31R

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12.158 Molecular Biogeochemistry Fall 2010

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