Direct NOE simulation from long MD trajectories.

A software package, MD2NOE, is presented which calculates Nuclear Overhauser Effect (NOE) build-up curves directly from molecular dynamics (MD) trajectories. It differs from traditional approaches in that it calculates correlation functions directly from the trajectory instead of extracting inverse sixth power distance terms as an intermediate step in calculating NOEs. This is particularly important for molecules that sample conformational states on a timescale similar to molecular reorientation. The package is tested on sucrose and results are shown to differ in small but significant ways from those calculated using an inverse sixth power assumption. Results are also compared to experiment and found to be in reasonable agreement despite an expected underestimation of water viscosity by the water model selected.

Probing alanine transaminase catalysis with hyperpolarized 13CD3-pyruvate.

Hyperpolarized metabolites offer a tremendous sensitivity advantage (>10(4) fold) when measuring flux and enzyme activity in living tissues by magnetic resonance methods. These sensitivity gains can also be applied to mechanistic studies that impose time and metabolite concentration limitations. Here we explore the use of hyperpolarization by dissolution dynamic nuclear polarization (DNP) in mechanistic studies of alanine transaminase (ALT), a well-established biomarker of liver disease and cancer that converts pyruvate to alanine using glutamate as a nitrogen donor. A specific deuterated, (13)C-enriched analog of pyruvic acid, (13)C3D(3)-pyruvic acid, is demonstrated to have advantages in terms of detection by both direct (13)C observation and indirect observation through methyl protons introduced by ALT-catalyzed H-D exchange. Exchange on injecting hyperpolarized (13)C3D(3)-pyruvate into ALT dissolved in buffered (1)H(2)O, combined with an experimental approach to measure proton incorporation, provided information on mechanistic details of transaminase action on a 1.5s timescale. ALT introduced, on average, 0.8 new protons into the methyl group of the alanine produced, indicating the presence of an off-pathway enamine intermediate. The opportunities for exploiting mechanism-dependent molecular signatures as well as indirect detection of hyperpolarized (13)C3-pyruvate and products in imaging applications are discussed.

Exchange facilitated indirect detection of hyperpolarized 15ND2-amido-glutamine.

Hyperpolarization greatly enhances opportunities to observe in vivo metabolic processes in real time. Accessible timescales are, however, limited by nuclear spin relaxation times, and sensitivity is limited by magnetogyric ratios of observed nuclei. The majority of applications to date have involved direct (13)C observation of metabolites with non-protonated carbons at sites of interest ((13)C enriched carbonyls, for example), a choice that extends relaxation times and yields moderate sensitivity. Interest in (15)N containing metabolites is equally high but non-protonated sites are rare and direct (15)N observation insensitive. Here an approach is demonstrated that extends applications to protonated (15)N sites with high sensitivity. The normally short relaxation times are lengthened by initially replacing protons (H) with deuterons (D) and low sensitivity detection of (15)N is avoided by indirect detection through protons reintroduced by H/D exchange. A pulse sequence is presented that periodically samples (15)N polarization at newly protonated sites by INEPT transfer to protons while returning (15)N magnetization of deuterated sites to the +Z axis to preserve polarization for subsequent samplings. Applications to (15)ND(2)-amido-glutamine are chosen for illustration. Glutamine is an important regulator and a direct donor of nitrogen in cellular metabolism. Potential application to in vivo observation is discussed.

Rhizobium meliloti produces a family of sulfated lipooligosaccharides exhibiting different degrees of plant host specificity.

We have shown that a Rhizobium meliloti strain overexpressing nodulation genes excreted high amounts of a family of N-acylated and 6-O-sulfated N-acetyl-beta-1,4-D-glucosamine penta-, tetra-, and trisaccharide Nod factors. Either a C(16:2) or a C(16:3) acyl chain is attached to the nonreducing end subunit, whereas the sulfate group is bound to the reducing glucosamine. One of the tetrasaccharides is identical to the previously described NodRm-1 factor. The two pentasaccharides as well as NodRm-1 were purified and tested for biological activity. In the root hair deformation assay the pentasaccharides show similar activities on the host plants Medicago sativa and Melilotus albus and on the non-host plant Vicia sativa at a dilution of up to 0.01-0.001 microM, in contrast to NodRm-1, which displays a much higher specific activity for Medicago and Melilotus than for Vicia. The active concentration range of the pentasaccharides is more narrow on Medicago than on Melilotus and Vicia. In addition to root hair deformation, the different Nod factors were shown to induce nodule formation on M. sativa. We suggest that the production of a series of active signal molecules with different degrees of specificity might be important in controlling the symbiosis of R. meliloti with several different host plants or under different environmental conditions.

Formation of disaccharides related to heparin and heparan sulfate by chemical modification of maltose.

Maltose has been converted into 4-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-L-idopyranuronic acid, 4-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-D-glucopyranose, and 4-O-alpha-D-glucopyranosyl-L-idopyranose, the first two disaccharides being related structurally to biose sequences in heparin and heparan sulfate. Used as the starting material was a major product of the kinetic acetonation of maltose, namely, 2,3:5,6-di-O-isopropylidene-4-O-(4,6-O-isopropylidene-alpha-D-glucopyran osyl)-aldehydo-D-glucose dimethyl acetal. It was subjected to a sequence of transformation that included the introduction of a 2'-amino-2'-deoxy function into the glucosyl group, the inversion of C-5 in the glucose residue to give the L-ido configuration, oxidation at position 6, and cyclisation of the acyclic dimethyl acetal to give the desired pyranuronic acid. In the formation of the latter, the 5-O-levulinoyl substituent was found to be less prone to acyl migration to O-6 than more conventional ester groups. The relative acid labilities of the O-isopropylidene and dimethyl acetal groups are compared, and conformations of the acyclic residues of some disaccharide derivatives are discussed.