Organic Pedagogical Electronic Network

Isotopic Labeling

Kristina Melnik & Stephanie FeltenUniversity of Utah

Isotopic Labeling

References: http://en.wikipedia.org/wiki/Isotopic_labeling, http://en.wikipedia.org/wiki/Crossover_experiment_%28chemistry%29,http://www.chemgapedia.de/vsengine/vlu/vsc/de/ch/12/oc/vlu_organik/aufklaerung/aufklaerung.vlu/Page/vsc/de/ch/12/oc/aufklaerung/a6_isotopenmarkierung2/a6isotopenmarkierung2.vscml.html

Overview: Technique that is used to elucidate mechanism of a reaction or in a metabolic pathway and in a cell. The labeling takes place by exchanging a specific atom with their isotope. The detecting of the isotopes in the product helps to understand the possible mechanism and the stereochemistry in this sequence of the reaction. The detection of the isotopic labels is dependent on the kind of isotope. Radioactive isotopes like 3H 14C are measured radiochemical. Stable isotopes like 2H and 13C are detected for example with NMR- and IR-spectroscopy.

Elucidation of Mechanism

R1 OH

OH18O R2+

R1 O R2R118O R2

O OH2O + H2

18O+

O

O

OH OH

OH OH

Isotopic label experiment Crossover experiment

Intramolecular (non crossover)

Intermolecular (crossover)

Isotopic LabelingOverview: Technique that is used to elucidate mechanism of a reaction or in a metabolic pathway and in a cell. The labeling takes place by exchanging a specific atom with their isotope. The detecting of the isotopes in the product helps to understand the possible mechanism and the stereochemistry in this sequence of the reaction. The detection of the isotopic labels is dependent on the kind of isotope. Radioactive isotopes like 3H 14C are measured radiochemical. Stable isotopes like 2H and 13C are detected for example with NMR- and IR-spectroscopy.

Elucidation of Stereochemistry

OH

HH

Hpro-R

pro-S

References: http://en.wikipedia.org/wiki/Isotopic_labeling, http://en.wikipedia.org/wiki/Crossover_experiment_%28chemistry%29.Citron, C. A.; Brock, N. L.; Tudzynski, B.; Dickschat, J. S., Chem. Commun., 2014, 50, 5224-5226.

Application of Isotopic labeling in metabolic pathway

References: Brock, N. L.; Mencke, M.; Klapschinski, T. ; Dickschat, J. S., Org. Biomol. Chem., 2014, 12, 4318-4323; Brock, N. L.; Citron, C. A.; Zell,C.;Berger, M.; Wagner-Doebler, I.; Petersen, J.; Brinkhoff, T.; Simon, M.; Dickschat, J. S., Beilstein J. Org. Chem., 2013, 9, 942-950.

Case Study 1: Sulfur volatiles production from the Roseobacter cladeDimethylsulfoniopropionate (DMSP) is one of the most important organic sulfur metabolite in marine environment and is degraded by marine bacteria in two competing pathways. Demethylation Pathway forms methanethiol (MeSH), while the Cleavage Pathway forms dimethylsulfide (DMS). For the distinct conclusion on the sulfur source for the volatile sulfur compounds, [ 34S]DMSP was synthesized and used in a feeding experiment.

Demethylation Pathway 34S O

FH4- Me-FH4 34S

O

OH 34S

O

SCoA

34S

O

SCoAO

ATP,HSCoA

- AMP - P2O7

4-

FAD- FADH2

NAD+,2 H2O

- HSCoA- Me34SH- CO2

- NADH

O

DMSP

Cleavage Pathway - DM34S

O

OHHO

O

OH - DM34SH2O

34S O

O

DMSP

The sulfur volatiles emitted by the bacteria were analysed by GC-MS and the data revealed that DMSP is efficiently degraded to MeSH via Demethylation pathway.

Application of Isotopic labeling in metabolic pathway

References: Citron, C. A.; Brock, N. L.; Tudzynski, B.; Dickschat, J. S., Chem. Commun., 2014, 50, 5224-5226.

Case Study 2: Biosynthesis of diterpenoids in Fusarium fujikuroia. elucidation of ring construction mechanism

H

H

O

HO

OH

H

CO2HH

OC

H

HO2C COHH

1 2 3 4

5

H

HHO2C

H

HHO2C

OH

ring construction

H

HHO2C H6

H7

H7H6 O Fe

H

HHO2C H6

H7

H6 HO Fe

H

HHO2C H6

H7

OHH6 O Fe

H

HHO2C H6

H7

O HHO Fe

H

CH7

HHO2C H6

2 3

4 isotopic label

Application of Isotopic labeling in metabolic pathway

References: Citron, C. A.; Brock, N. L.; Tudzynski, B.; Dickschat, J. S., Chem. Commun., 2014, 50, 5224-5226.

Case Study 2: Biosynthesis of diterpenoids in Fusarium fujikuroib. elucidation of conversion

OO

OH

H

H

O

HO

OH

H

CO2H

H

OC

6 1 5 isotopic label

O

HO

OH

H

CO2H

H

OC

O

HO

OH

H

CO2HH

OC

Problems

1. Take a look at the following reaction and tell which experiment is useful to show the obtained products.

Hint: The mechanism for this reaction is a benzidine rearrangement!

NHHN

ROOR

+

NHHN

ROOR

H ?

Problems2. The reaction of dimethylallyldiphosphate (DMAPP) with isopentyldiphosphate (IPP) to geranyldiphosphate(GPP) is an important enzyme catalyzed reaction in the terpene metabolism. (OPP = diphosphate, P2O7

3-).Analysis of the stereochemical process shows:

– Nucleophilic attack on DMAPP at position 1 (OPP is the leaving group).The reaction proceeds under inversion of the configuration at C1.

– The nucleophile is the 4th position of IPP, so that it attacks from the Si-site in position 4. (Remeber Rule: Z>E for the priorities of the two hydrogens!)

– At the same time the deprotonation at IPP takes place at position 2. The 2-pro-R-proton is abstracted.

This stereochemical process can be examined with isotopic labeling. Show the mechanism of the stereochemical synthesis of GPP with isotopic labeled starting materials. What kind of isotopic label would you take at which position?

OPP

DMAPP

1OPP

H4

2

IPP

OPP

GPP

Solutions

References: http://www.chemgapedia.de/vsengine/vlu/vsc/en/ch/12/oc/vlu_organik/aufklaerung/aufklaerung_e_kreuz_kinetik.vlu.htmlShine, H.J.; Zmuda, H.: Park, K.H.; Kwart, H.; Horgan, A.G.; Brechbiel, M. J. Am. Chem. Soc., 1982, 104, 2501-2509.Laue, A.; Plagens, A., Namen- und Schlagwort-Reaktionen, 5. Auflage, Teubner, Wiesbaden 2006, S. 40.

1) Two products are obtained in the reaction which means that it is a intramolecular process. Only symmetrically substituted benzidines are obtained. The experiment to distinguish the products is the Crossover experiment.

The products are:

The mechanism is shown for product 1:

MeO OMe

H2N NH2

EtO OEt

H2N NH2+

NHHN

MeOOMe

N NH

HH

H

H

MeO OMe

MeO OMe

H2N NH2 - 2H

MeO OMe

H2N NH2

Solutions2) Labeling of the starting materials would give the following structures:

References: Dickschat, J. S.; old exam question, wintersemester 2012/2013, TU Brunswick, Germany.

OPP

DMAPP

1OPP

H4

2

IPP

OPP

GPP

OPP

DHOPP

DHD

HOPP

H D

D H

The explanation for the reaction is the following:

OPP

DH

Inversion

OPPDHD

H

Si-attackpro S

pro R

This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.

Contributed by: Kristina Melnik & Stephanie Felten (Undergraduate Students)

University of Utah

2014