Thomas E. Walker

Photosynthetic preparation and characterization of 13C-labeled carbohydrates in agmenellum quadruplicatum

Abstract The blue-green alga Agmenellum quadruplicatum (strain PR6) has been used to prepare photobiosynthetically 13 C-labeled d -glucose, 2- O -(α- d -glucopyranosyl)-glyceric acid (glucosylglycerate), 2-hydroxy-1-(hydroxymethyl)ethyl α- d -gluco-pyranoside (glucosylglycerol), and α- d -glueopyranosyl β- d -fructofuranoside (sucrose). When grown to a cell density of 4.4 g.L -1 (dry weight) under nitrate-nitrogen limiting growth conditions for 120 h, the algal cells contained 38% of the dry-cell weight as(1 → 4)-α- d -glucan (amylose). About 1% of the dry-cell weight was glucosylglycerol, glucosylglycerate, and sucrose. Glutamate was obtained, together with carbohydrates of low molecular weight, when the cells were extracted with chloroform-methanol; d -glucose was recovered from the extracted cells by acid hydrolysis of the starch. The algae were grown by using 20 mol% [ 13 C] carbon dioxide for preparation of labeled carbohydrates and for cellular component identification by whole-cell n.m.r. spectroscopy.

A 13C nuclear magnetic resonance study of the interaction of ligands with arginine residues in dihydrofolate reductase

/sup 13/C NMR spectra of Streptococcus faecium dihydrofolate reductase containing (/sup 13/C-guanidino) arginine and ligand complexes with the labeled enzyme are reported. The spectrum of the native enzyme shows 5 well-resolved resonances (the enzyme contains 8 Arg) with a chemical shift range of 1.2 ppM. Addition of ligands to the enzyme produces distinct changes in the enzyme spectrum, and demonstrates that /sup 13/C NMR of labeled protein can be used in studies of protein-ligand interactions. An example of the use of /sup 13/C-depleted material is also presented.

Proton relaxation in 13C-enriched molecules; 13C T1 and NOE data from proton magnetic resonance measurements☆

The proton relaxation of several 13C-labeled molecules has been studied. A comparison of the spin-lattice relaxation rate of the center proton resonances corresponding to the unlabeled molecule with the relaxation rates of the 13C satellites enables the contribution of the 13C1H dipolar interaction to be accurately determined. This interaction is roughly equivalent in magnitude to the 1H1H dipolar interaction for a methylene group due to the smaller 13C1H distance. The data can be used to obtain reasonable values for the T113C, of protonated carbons. A comparison with the directly measured T113c values enables a quick determination of the nuclear Overhauser enhancement to be made.

The Synthesis of Carbon-13 Enriched Monosaccharides Derived from Glucose and Mannose

Abstract A modified Kiliani-Fischer reaction is used to prepare multigram quantities of [1-13C]-enriched glucose and mannose which are converted chemically or enzymatically into other labeled monosaccharides. The simplest conversion is the synthesis of labeled fructose from labeled glucose using commercially available immobilized glucose isomerase. The equilibrium for this reaction provides a 1:l mixture of glucose and fructose which can be separated by chromatography. The equilibrium can be shifted toward fructose by treating the reaction with germanate ion. [1-13C]Mannose can be converted into more useful sugars using a modification of the Lobry de Bruyn-Alberda van Ekenstein transformation. In this reaction, D-[1-13C]mannose is treated with an aqueous solution of dilute alkali and phenylboronate to form a mixture of labeled fructose, mannose and glucose. Fructose can be converted to a mixture of methyl fructofuranosides by using trifluoroacetic acid in methanol. [2-13C]Dihydroxyacetone can be prepared ...

Application of doubly decoupled 13C {1H, 14N}NMR spectroscopy to studies of the conformation and dynamics of the choline headgroup of phospholipids

The high symmetry about the choline nitrogen atom leads to resolvable scalar 13C–14N coupling in the 13C NMR spectrum of choline. For membranes or model membrane systems containing phosphatidyl choline molecules, this coupling can be partially or completely collapsed due to the slower motion and consequently shorter 14N spin lattice relaxation time of the choline nitrogen. In the present study, this effect has been probed by comparison of the 13C NMR spectra obtained using conventional proton decoupling with spectra obtained using simultaneous 14N and 1H decoupling,i.e., 13C {1H, 14N}nmR spectroscopy. The results indicate a partially collapsed triplet structure for the choline methyl resonances in several sonicated lipid vesicles as well as in whole Chinese Hamster Ovary cells labeled with a 3 : 3 : 1 mixture of [1-13C]-, [2-13C]-, and [13CH3] choline. Further, the sharpening of the choline C-2 resonance resulting from the 14N decoupling makes possible the observation of three bond 13C–31P scalar coupling and, consequently, a conformational analysis of the rotational isomerism about the choline OC-1 bond.

Synthesis of 2-deoxy-d-arabino-(6-13C)hexose

2-Deoxy-D-arabino-[6-13C]hexose (10), to be used to test the stability of 2-deoxy-D-arabino-hexose 6-phosphate in brain tissue, was prepared. 2-Deoxy-D-arabino-hexose was labeled at C-6 because of the large difference in chemical shift between C-6 in the free sugar and C-6 in the 6-phosphate. The synthetic scheme resembled that used for the synthesis of D-[6-13C]glucose that involved the removal of C-6 from D-glucose followed by its replacement with 13C. The protected derivative methyl 2-deoxy-alpha-D-arabino-hexofuranoside was prepared, using trifluoroacetic acid in methanol. This was treated with periodate, which cleaves only between C-5 and C-6, to afford an aldehyde which reacted directly with K13CN to give a mixture of the D-arabino and L-xylo nitriles. The enriched nitriles were reduced with hydrogen in the presence of 5% Pd-carbon catalyst to a mixture of 6-aldehydo sugars. These were reduced with NaBH4 to a mixture of the two labeled methyl furanosides. Acid hydrolysis followed by ion-exchange chromatography on AG-50(Ca2+) resin at 65 degrees gave 10 in an overall yield of 16% from K13CN.

Biosynthesis of trehalose byBrevibacterium flavum: Use of long range13C−13C coupling data to characterize triose phosphate isomerase activity

The13C isotopic labeling pattern in the disaccharide trehalose (l,1-α-α-D-glucose) produced by the micro-organismBrevibacterium flavum when grown on a medium containing [1-{au13}C]glucose has been determined. Long range coupling between C-1 and C-6 carbons of the glucose units can be observed in the excreted material . It is proposed that some of the13C isotopomers in the excreted trehalose reflect the labeling pattern in (unobserved) fructose 1,6-diphosphate. Analysis of the label distribution within the framework of a steady state kinetic model allows an analysis of the contributions of the hexose monophosphate shunt and the degree of equilibration of triose phosphate isomerase. Analogous measurements on excreted glucose could be carried out in other organisms.