The association constants for the interactions of 2-hydroxy-4-methoxyacetophenone, 2-hydroxy-5-methoxyacetophenone, 2-hydroxy-6-methoxyacetophenone, 3-hydroxy-4-methoxyacetophenone and 4-hydroxy-3-methoxyacetophenone with β-cyclodextrin in water were measured by (1)H NMR and by isothermal titration calorimetry. Very good agreement was obtained between the different methods. The errors associated with the NMR method for measuring mM binding affinities were estimated to be 10-30%, and by isothermal titration calorimetry, 10-20%. Rotating frame nuclear Overhauser effect spectroscopy studies show that the solution phase host-guest complexes formed by β-cyclodextrin with these hydroxymethoxyacetophenone derivatives are not structurally well defined but that the hydroxymethoxyacetophenone derivatives are mostly associated with the narrow primary hydroxyl rim.

The discovery of a novel series of 8-azabicyclo[3.2.1]octan-3-yl)-3-(4-chlorophenyl) propanamide antagonists of the vasopressin V(1A) receptor is disclosed. Compounds 47 and 48 were found to be high affinity, selective vasopressin V(1A) antagonists.

Knowledge of the three-dimensional structure of a ligand in the binding site of its biological receptor is a valuable asset that can assist disease research and guide drug discovery. Solid-state nuclear magnetic resonance (SSNMR) is a useful high-resolution technique for the structural analysis of small molecule or peptide ligands when bound to receptors. SSNMR-derived constraints on the molecular conformations of isotopically (e.g., (13)C and (15)N) enriched ligands usually take the form of through-space distances between atomic nuclei that are separated by three or more bonds. It is advantageous to supplement such distance measurements with independent geometric constraints to resolve structural ambiguities arising from molecular symmetry. Here it is demonstrated that multiple torsional angle constraints can be measured directly for a uniformly labelled biological ligand at a realistically low concentration (150 nmoles) in a practicable experiment time. A simple adaptation of a standard one-dimensional (13)C double-quantum filtered SSNMR experiment is used to measure the relative orientations of C-H bonds in CH(2)-CH and CH(2)-CH(2) groups, which influence (13)C double quantum signal amplitudes in a predictable way. The methodology is applied to uniformly (13)C and (15)N labelled glutamate ([U-(13)C,(15)N]Glu) bound to the ligand binding domain of the ionotropic glutamate receptor 2 (GluR2) in a microcrystalline preparation. Two torsional angle constraints are sufficient to eliminate the structural ambiguities associated with (13)C-(15)N interatomic distance measurements, and thus provide a reliable representation of the conformation of glutamate in its receptor-bound state.

This article is a review with 83 references of the application of NMR to the measurement of the dissociation constants of protein-ligand complexes. After briefly discussing some general concepts of molecular stability, the text turns to consider which NMR parameters are reporters of complex formation. The available data treatments required to translate observed NMR effects into quantitative measurements of the stability of the complex in the form of the dissociation constant (KD) are introduced. Linearisation methods and curve fitting methods are explained in detail and are illustrated with examples drawn from recent reports of protein-small molecule interactions. Throughout the text examples of the commonly observed NMR parameters , 1 / T1 and 1 / T2 are drawn from biological studies of 1H, 31P, 19F 15N (and other nuclei). The advantages of NMR diffusion experiments as a measure of KD are considered. Some less frequently used NMR approaches, some new ideas and some non-general methods are grouped together in a miscellaneous section. The major sources of errors in the determination of KD are identified. This allows recommendations for optimal experimental set up. Options for dealing with strong binding are reviewed. Finally, the implications of abstracting KD data from high throughput screening experiments are considered and several different approaches to generate this data are discussed.

The solution-state NMR spectra of a per-6-substituted gamma-cyclodextrin show some interesting dynamic properties. At high temperature (353 K), the (1)H NMR spectrum shows dynamic averaging of the different conformations. This averaging is no longer observed on cooling of the cyclodextrin solution to 278 K, resulting in NMR spectra with a large (1)H and (13)C chemical shift dispersion. The complete assignment of the eight unique glucosyl residues was achieved using COSY, HSQC and exchange spectroscopy. A ROESY spectrum, with a short mixing time to reduce the effects of exchange, gives correlations that lead to the determination of the connectivity of all eight glucosyl residues. On the NMR time-scale, the cyclodextrin is highly dynamic; the lower temperature minimum energy conformation has one of the aromatic rings self-complexed and a distorted cyclodextrin torus.

The usefulness of bovine serum albumin (BSA) as a model protein for testing NMR methods for the study of protein-ligand interactions is discussed. Isothermal titration calorimetry established the binding affinity and stoichiometry of the specific binding site for L-tryptophan, D-tryptophan, naproxen, ibuprofen, salicylic acid and warfarin. The binding affinities of the same ligands determined by NMR methods are universally weaker (larger KD). This is because the NMR methods are susceptible to interference from additional non-specific binding. The L-tryptophan-BSA and naproxen-BSA systems were the best behaved model systems.

The binding of rocuronium bromide to 6-perdeoxy-6-per(4-carboxyphenyl) thio-gamma-cyclodextrin sodium salt, displays biphasic behaviour characteristic of the formation of a binary and 2 : 1 ternary guest-host complex in aqueous solution. Thermodynamic and structural data on this sequential complexation process can be rationalised within a single model involving switching of the conformational equilibria of both the rocuronium bromide and cyclodextrin molecules. Isothermal titration calorimetry (ITC), NMR and. fluorescence experiments in solution, together with X-ray crystallography and molecular modelling, suggest that in order to induce encapsulation both rocuronium bromide and the modified cyclodextrin undergo conformational changes. Ring A of rocuronium bromide `switches' from the more sterically encumbered chair to the sterically less demanding twist-boat, whilst the modified cyclodextrin "opens" its cavity to allow the steroid to enter. The recognition and mutual induced fit between cyclodextrin and steroid represents a classic example of dynamic host-guest chemistry.

A collection of small molecules (MW < 350 Da) was screened for binding to human factor Xa using saturation transfer difference NMR spectroscopy to detect binding. The NMR screening experiments identified four hits. Binding isotherms constructed from NMR linewidth data showed that the binding affinities of the hits were all in the 30-210 microM range. Competition binding experiments showed that three of the ligands were displaced by a known microM inhibitor of factor Xa. The success of the method for identifying new ligands and the relevance of this information to the design of new factor Xa inhibitors are discussed.

The interaction of Rocuronium Bromide, and a model steroid Org 7402, with three cyclodextrins (β‐cyclodextrin, γ‐cyclodextrin and Org 25969) was studied by solution state NMR experiments. Stoichiometries and binding constants were determined from 1H chemical shift titrations. All of the systems formed 1 : 1 complexes. Most of the complexes were in fast exchange with unbound species on the NMR time scale, but the most tightly bound complex (Rocuronium Bromide–Org 25969) was in the slow exchange regime. The geometry of the complexes was inferred from 1H and 13C NMR shift changes upon complexation and from intramolecular NOE correlations. Rocuronium Bromide forms a weak complex with β‐cyclodextrin (Ka = 3.3 ± 0.5 × 103 M−1) and no clear picture of the structure of the complex emerges. The complexes with γ‐cyclodextrin (Ka = 1.8 ± 0.2 × 104 M−1) and Org 25969 (Ka > 105 M−1) are true inclusion complexes with the steroid located inside the central void of the cyclodextrin. Copyright © 2002 John Wiley & Sons, Ltd.

A series of carboxyl-containing cyclophanes have been designed and synthesised as chemical chelators (or host molecules) of cationic muscle relaxant drugs (or guest molecules). Three of these cyclophane derivatives, 1-3, have been shown by NMR to form 1:1 complexes with the muscle relaxants pancuronium, and gallamine, in D(2)O, with association constants up to 10(4) M(-1). When tested in an in vitro chick biventer muscle preparation, the cyclophanes reversed the neuromuscular block induced by pancuronium and gallamine, with having the most effective reversal against pancuronium (EC(50) 40 microM.

A series of per-6-substituted cyclodextrin derivatives was synthesized as synthetic host molecules for rocuronium, a steroidal muscle relaxant. By forming host-guest complexes with rocuronium, these cyclodextrin derivatives reverse the muscle relaxation induced by rocuronium in vitro and in vivo. The isothermal microcalorimetry data are consistent with the biological data supporting the encapsulation mechanism of action. Binary and biphasic complexes are reported with NMR experiments clearly showing free and bound rocuronium. [structure: see text]

The complexes formed by the steroid rocuronium bromide with four different cyclodextrins (β‐cyclodextrin, γ‐cyclodextrin, Org 26054 and Org 25969) were investigated. The diffusion coefficients of the steroid–cyclodextrin complexes were 6–15% lower than those of the native cyclodextrins, consistent with a 1 : 1 stoichiometry for all of the complexes. Copyright © 2002 John Wiley & Sons, Ltd.

The complexes of cyclohexylacetic acid and cholic acid with beta -cyclodextrin were studied by NMR diffusion coefficient measurements. The diffusion coefficient of the 1:1 cyclohexylacetic acid/beta -cyclodextrin complex, K-a = 1800 +/- 100 M-1, is slightly slower (3.23 +/- 0.07 x 10(-6) cm(2) s(-1)) than that of beta -cyclodextrin (3.29 +/- 0.07 x 10(-6) cm(2) s(-1)). The diffusion coefficient of the 1:1 cholic acid/beta -cyclodextrin complex, K-a 5900 +/- 800 M-1, is significantly slower (2.93 +/- 0.07 x 10(-6) cm(2) s(-1)) than that of beta -cyclodextrin. The results indicate that caution should be exercised when studying host-guest complexation by the so-called 'single point' technique. A novel data treatment is introduced which takes into account the diffusion behavior of all of the species when determining K-a. Experimentally determined diffusion coefficients of complexes are also a useful probe of the size of host-guest complexes.

Cameron, K. S.; Fletcher, D.; Fielding, L.; Clark, J. K.*; Zhang, M.-Q.*; Orbons, L. P. M. Author Information

Six steroids with inverted stereochemistry at C-13 (13-epi) were studied. Measurement of vicinal proton-proton couplings was facilitated by a 1D TOCSY experiment, which provided efficient deconvolution of the ring D protons from other signals. A simple semi-quantitative analysis of the ring D couplings was found to be sufficient to determine the conformation of ring D. Examples of 13 alpha, 14 beta half-chair, 17 beta envelope and 16 beta envelope conformations were found. Copyright (C) 2001 John Wiley & Sons, Ltd.

Various cyclic ether and other 3 alpha-hydroxyandrostane derivatives bearing a conformationally constrained hydrogen-bonding moiety were prepared. Their anesthetic potency and their binding affinity for GABA(A) receptors, measured by intravenous administration to mice and inhibition of [(35)S]TBPS binding to rat whole brain membranes, were compared with that of known anesthetic 3 alpha-hydroxypregnan-20-ones. Synthetic steroids with similar in vitro and in vivo activities to the endogenous 3 alpha-hydroxypregnan-20-ones all had an ether oxygen on the beta-face of the steroid D-ring. These results suggest that for optimal GABA(A) receptor modulation, the hydrogen bond-accepting substituent should be near perpendicular to the plane of the D-ring on the beta-face of the steroid.

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Molecular beacons are important bioanalytical probes most often constructed from a single-stranded oligonucleotide which has been labeled at opposite termini with a fluorophore and a quencher. When the fluorophore and quencher are in close proximity, ideally no fluorescence is observed due to Fluorescence Resonance Energy Transfer (FRET). 4-Dimethylaminoazobenzene-4′-carboxylic acid (DABCYL) is a commonly used quencher in molecular beacons; however, DABCYL is unable to form a base-pair and is conventionally installed as an overhanging residue. In this arrangement, the DABCYL moiety has substantial mobility which affects the accuracy of FRET-related distance/orientation measurements and also limits the types of other conjugates that can be prepared. To address these limitations, we have synthesized peptide nucleic acid (PNA) analogues possessing DMPAU (5-[(4-dimethylaminophenyl)diazenyl]uracil) and NPhpC (6-(4-nitrophenyl)pyrrolocytosine) nucleobases which act as dark fluorophores, i.e. quenchers, while retaining the ability to base pair by canonical hydrogen bonding. This will allow more flexible design and construction of molecular beacons based on the insertion of nucleobase fluorophores and quenchers into the stem sequence. We have quantified the quenching ability of DMPAU and NPhpC PNA analogues by determining the Stern-Volmer constants against the intrinsically fluorescent nucleobase, phenylpyrrolocytosine, and other blue emitting fluorophores (pyrene, acridone). The hydrogen bonding ability of DMPAU with adenine, as determined by 1H NMR titration, is relatively unperturbed compared to uracil and thymine.

The 1H and 13C NMR spectra of the steroidal neuromuscular blocking drugs pancuronium bromide, vecuronium bromide, rocuronium bromide and Org. 9487 are presented. The 13C NMR spectra are fully assigned. NOE data and variable-temperature studies show that there is considerable conformational freedom in both the ring A substituents and the sterically more crowded ring D substituents. The barrier to nitrogen-ring inversion in the non-quaternized piperidine ring (ΔG≠) is ca. 10–11 kcal mol-1. The quaternized ring D piperidine (or pyrollidine) rings are free to rotate with respect to the steroid. The vicinal couplings 13CO–O–C–1H, ca. 4 Hz, suggest that the ester moieties have similar rotational freedom to simple esters. © 1998 John Wiley & Sons, Ltd.