Isotopic labeling

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.

Kristina Melnik & Stephanie Felten (Undergraduate Students)
University of Utah
2014

1. Isotopic Labeling Kristina Melnik & Stephanie Felten University of Utah

2. 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 3 H 14 C are measured radiochemical. Stable isotopes like 2 H and 13 C are detected for example with NMR- and IR-spectroscopy. Elucidation of Mechanism Isotopic label experiment Crossover experiment Intramolecular (non crossover ) Intermolecular ( crossover )

3. Isotopic Labeling 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 3 H 14 C are measured radiochemical. Stable isotopes like 2 H and 13 C are detected for example with NMR- and IR-spectroscopy. Elucidation of Stereochemistry 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.

4. 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 clade Dimethylsulfoniopropionate (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, [ 34 S]DMSP was synthesized and used in a feeding experiment. Demethylation Pathway Cleavage Pathway 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.

5. 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 fujikuroi a. elucidation of ring construction mechanism r ing construction

6. 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 fujikuroi b. elucidation of conversion

7. Problems 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!

8. Problems 2 . The reaction of dimethylallyldiphosphate (DMAPP) with isopentyldiphosphate (IPP) to geranyldiphosphate (GPP) is an important enzyme catalyzed reaction in the terpene metabolism. (OPP = diphosphate , P 2 O 7 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 4 th 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?

9. Solutions References: http://www.chemgapedia.de/vsengine/vlu/vsc/en/ch/12/oc/vlu_organik/aufklaerung/aufklaerung_e_kreuz_kinetik.vlu.html Shine, 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 :

10. Solutions 2) Labeling of the starting materials would give the following structures : References: Dickschat, J. S.; old exam question, wintersemester 2012/2013, TU Brunswick, Germany. The explanation for the reaction is the following:

11. Contributed by: Kristina Melnik & Stephanie Felten (Undergraduate Students) University of Utah 2014