The absolute sign of J coupling constants determined using the order matrix calculation.

A novel methodology using the order matrix calculation to determine the absolute sign of spin-spin couplings based on the structure of organic compounds is presented. The sign of the residual dipolar coupling (RDC) depends on the sign of corresponding scalar spin-spin coupling constant and the sign of the RDC has a dramatic influence on the order matrix calculation. Therefore, the sign of the spin-spin coupling constant can be obtained by an order matrix calculation through the corresponding RDC. Six types of spin-spin coupling constants, including 2J(H,H), 1J(C,F), 2J(C,F), 3J(C,F), 2J(F,H) and 3J(F,H), were obtained simultaneously. Except for 3J(C,F) where the measured RDCs have very small magnitudes, the signs were determined unambiguously.

Complete relative stereochemistry of multiple stereocenters using only residual dipolar couplings.

Residual dipolar couplings (RDCs), in combination with molecular order matrix calculations, were used to unambiguously determine the complete relative stereochemistry of an organic compound with five stereocenters. Three simple one-dimensional experiments were utilized for the measurements of (13)C-(1)H, (13)C-(19)F, (19)F-(1)H, and (1)H-(1)H RDCs. The order matrix calculation was performed on each chiral isomer independently. The fits were evaluated by the comparison of the root-mean-square deviation (rmsd) of calculated and measured RDCs. The order tensor simulations based on two different sets of RDC data collected with phage and bicelles are consistent. The resulting stereochemical assignments of the stereocenters obtained from using only RDCs are in perfect agreement with those obtained from the single-crystal X-ray structure. Six RDCs are found to be necessary to run the simulation, and seven are the minimum to get an acceptable result for the investigated compound. It was also shown that (13)C-(1)H and (1)H-(1)H RDCs, which are the easiest to measure, are also the most important and information-rich data for the order matrix calculation. The effect of each RDC on the calculation depends on the location of the corresponding vector in the structure. The direct RDC of a stereocenter is important to the configuration determination, but the configuration of stereocenters devoid of protons can also be obtained from analysis of nearby RDCs.

RAMPED-UP NMR: multiplexed NMR-based screening for drug discovery.

The crucial step in drug discovery is the identification of a lead compound from a vast chemical library by any number of screening techniques. NMR-based screening has the advantage of directly detecting binding of a compound to the target. The spectra resulting from these screens can also be very complex and difficult to analyze, making this an inefficient process. We present here a method, RAMPED-UP NMR, (Rapid Analysis and Multiplexing of Experimentally Discriminated Uniquely Labeled Proteins using NMR) which generates simple spectra which are easy to interpret and allows several proteins to be screened simultaneously. In this method, the proteins to be screened are uniquely labeled with one amino acid type. There are several benefits derived from this unique labeling strategy: the spectra are greatly simplified, resonances that are most likely to be affected by binding are the only ones observed, and peaks that yield little or no information upon binding are eliminated, allowing the analysis of multiple proteins easily and simultaneously. We demonstrate the ability of three different proteins to be analyzed simultaneously for binding to two different ligands. This method will have significant impact in the use of NMR spectroscopy for both the lead generation and lead optimization phases of drug discovery by its ability to increase screening throughput and the ability to examine selectivity. To the best of our knowledge, this is the first time in any format that multiple proteins can be screened in one tube.

The effect of relaxation on the epitope mapping by saturation transfer difference NMR.

The effect of longitudinal relaxation of ligand protons on saturation transfer difference (STD) was investigated by using a known binding system, dihydrofolate reductase and trimethoprim. The results indicate that T1 relaxation of ligand protons has a severe interference on the epitope map derived from a STD measurement. When the T1s of individual ligand protons are distinctly different, STD experiments may not give an accurate epitope map for the ligand-target interactions. Measuring the relaxation times prior to mapping is strongly advised. A saturation time shorter than T1s is suggested for improving the potential epitope map. Reduction in temperature was seen to enhance the saturation efficiency in small to medium size targets.

A novel method for the determination of stereochemistry in six-membered chairlike rings using residual dipolar couplings.

A novel method for the determination of the relative stereochemistry of six-membered chairlike ring molecules by residual dipolar couplings is presented. C-H residual dipolar couplings were used to investigate the relative stereochemistry of 4,6-O-ethylidene-d-glucopyranose. For this and similar systems it is not necessary to acquire redundant dipolar couplings and to calculate the orientation order tensor. The presented methodology is a paradigmatic leap for the determination of the relative stereochemistry or remote stereochemistry in this kind of fused ring system. Residual dipolar coupling data were collected by 1D and 2D direct-measurement heteronuclear multiple quantum coherence (HMQC) spectroscopy. It was demonstrated that direct measurement of HMQC was quick and accurate for small molecules at natural abundance.

ID NMR Methods in ligand-receptor interactions.

The drug discovery process often involves the screening of compound libraries to identify drug candidates capable of binding to target macromolecules. New approaches in biological and chemical research are driving a change in the pharmaceutical industry. Recent advances in NMR spectroscopy such as affinity NMR techniques, which detect binding of a small molecule with a "receptor", have been shown to be valuable tools to perform rapid screening of compounds for biological activity. These NMR observable events include using relaxation, chemical shift perturbations, translational diffusion, and magnetization transfer. These one dimensional NMR methods increase both the throughput of screening and yield crucial data on the mode of binding. The practical utility of these techniques will be described.

Epitope mapping of ligand-receptor interactions by diffusion NMR.

A novel method based on diffusion NMR for the epitope mapping of ligand binding is presented. The intermolecular NOE builds up during a long diffusion period and creates a deviation from the linearity. The ligand proton nearest the protein generates the strongest NOE from protein during the diffusion period and has the largest deviation. Therefore, this diffusion artifact can be used to characterize the ligand binding epitope. The concept was investigated using dihydrofolate reductase (DHFR) and its ligand trimethoprim (TMP), and the epitope map of TMP on DHFR generated with this method is in excellent agreement with the structural and dynamic studies by crystallography and NMR, as well as the medicinal chemistry results.