The SIR provided (2S)-N-(2'-O-benzyl-glycoloyl)bomane-10-,2-sultam 11,2- 13C]acetic acid; 20g Recently, a new pathway to isopentenyl pyrophosphate has been discovered which emanates from carbohydrate metabolism. To probe distribution of this new pathway in nature and to elucidate further intermediates in the pathway, we are presently synthesizing 1-deoxy-D-[2,4-13C2]xylulose. Initially, a small quantity for exploratory work will be prepared from ethyl bromo- [ I - I 3C] acetate and [2-13C]acetone, which has been developed by the UW group. In the course of this work it became evident that this route is not very efficient and thus unsuitable for the production of larger quantities of this precursor. A second route has therefore been designed by the SIR, which makes effective use of the labeled synthons they have developed. If experiments on ubiquinone formation in E. coli with the initially synthesized precursor sample demonstrate the feasibility of this approach, a larger quantity of the compound will then be synthsized by the SIR. This precursor will be fed to a variety of biological systems synthesizing isoprenoid compounds and the products will be analyzed by 13C-NMR for the incorporation of 13C and the appearance of 13C_13C coupling, resulting from the rearrangement of the precursor, in the appropriate positions. This will give an overview of the relative importance of the new pathway compared to the classical mevalonate pathway. Next, we will incubate the double labeled substrate with washed cells and cell-free extracts of E. coli directly in the NMR spectrometer and examine the incubations periodically by 1H-detected 13C-NMR, looking specifically for double-quantum transitions, for the appearance of new species in which the two 13C nuclei are connected to each other. Since virtually no other metabolic pathway can give rise to extensive 13C-13C coupling, this will be a very specific probe for the intermediates in the new pathway of isoprenoid biosynthesis. The time course of the appearance and disappearance of new species will indicate their relative position in the reaction sequence, and labeling of the precursor in additional positions will allow the assignment of structures to these intermediates. These in vivo experiments will require high sensitivity to detect multiquantum transitions from very small amounts of transient compounds. This can be achieved on the 750 MHz NMR spectrometer at the Univ. of Wash.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Biotechnology Resource Grants (P41)
Project #
Application #
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Los Alamos National Lab
Los Alamos
United States
Zip Code
Martinez, Rodolfo A; Glass, David R; Ortiz, Erick G et al. (2014) Synthesis of isotopically labeled 1,3-dithiane. J Labelled Comp Radiopharm 57:338-41
Martinez, Rodolfo A; Glass, David R; Ortiz, Erick G et al. (2013) Large-scale preparation of (13) C-labeled 2-(phenylthio)acetic acid and the corresponding labeled sulfoxides and sulfones. J Labelled Comp Radiopharm 56:31-5
Creager, Melinda S; Jenkins, Janelle E; Thagard-Yeaman, Leigh A et al. (2010) Solid-state NMR comparison of various spiders' dragline silk fiber. Biomacromolecules 11:2039-43
Jenkins, Janelle E; Creager, Melinda S; Lewis, Randolph V et al. (2010) Quantitative Correlation between the protein primary sequences and secondary structures in spider dragline silks. Biomacromolecules 11:192-200
Kim, Sun Hee; Aznar, Constantino; Brynda, Marcin et al. (2004) An EPR, ESEEM, structural NMR, and DFT study of a synthetic model for the covalently ring-linked tyrosine-histidine structure in the heme-copper oxidases. J Am Chem Soc 126:2328-38
Ollivault-Shiflett, Morgane; Kimball, David B; Silks, L A Pete (2004) Synthesis of chiral 13C,77Se-labeled selones. J Org Chem 69:5150-2
Schmidt, Bryan; Hillier, Warwick; McCracken, John et al. (2004) The use of stable isotopes and spectroscopy to investigate the energy transducing function of cytochrome c oxidase. Biochim Biophys Acta 1655:248-55
Schmidt, Bryan; McCracken, John; Ferguson-Miller, Shelagh (2003) A discrete water exit pathway in the membrane protein cytochrome c oxidase. Proc Natl Acad Sci U S A 100:15539-42
Gray, Chandele R; Sanz-Cervera, Juan F; Silks, Louis A et al. (2003) Studies on the biosynthesis of asperparaline A: origin of the spirosuccinimde ring system. J Am Chem Soc 125:14692-3
Van Dien, Stephen J; Strovas, Tim; Lidstrom, Mary E (2003) Quantification of central metabolic fluxes in the facultative methylotroph methylobacterium extorquens AM1 using 13C-label tracing and mass spectrometry. Biotechnol Bioeng 84:45-55

Showing the most recent 10 out of 72 publications