Ronald C. Crouch

Design and synthesis of novel protease inhibitors. Tripeptide α′,β′-epoxyketones as nanomolar inactivators of the proteasome

Abstract Tripeptide α′,β′-epoxyketones were prepared stereospecifically starting from Boc-[S]-phenylalanine. Diastereomer 5b inhibited the chymotrypsin-like activity of porcine endothelial cell derived proteasome at low nanomolar concentrations.

Selective inverse multiple bond analysis. A simple 1D experiment for the measurement of long-range heteronuclear coupling constants

The general concept of selective excitation is well established and the methodology has been utilized on numerous occasions in a wide range of applications. An interesting application of selective pulses has been in the development of selective analogues of two-dimensional NMR experiments. The first such effort germane to our work was that of Bax and Freeman ( 1)) in which they described the selective heteronuclear 2D J spectrum. Davis applied semiselective proton pulses to the Ha resonances of peptides (2) to establish long-range correlations to carbonyl resonances with high resolution in both frequency domains. In a paper published very shortly thereafter, Bermal and co-workers (3) described a group of two-dimensional pulse sequences intended to provide access to heteronuclear coupling constants that employed semiselective Gaussian 13C pulses. Kessler and colleagues have employed semiselective 13C pulses to improve the Fi resolution in proton-detected heteronuclear long-range correlation experiments (4). Supportive ancillary work has addressed the problem of extracting coupling constants from complex cross-peak multiplets observed in proton-detected heteronuclear correlation spectra (5, 6). The importance of measuring small heteronuclear coupling constants has not waned, as evidenced by recently reported heteronuclear two( 7, 8) and three-dimensional (9) studies designed to determine and utilize small heteronuclear coupling constants of biologically important molecules. In a departure from existing multidimensional methods, we report a simple, one-dimensional pulse sequence designed to allow the measurement of long-range heteronuclear coupling constants and to provide the means for establishing long-range heteronuclear connectivities. The earliest reports of one-dimensional analogs of two-dimensional NMR experiments were those of Berger ( IO, II), which were followed by the work of Kessler and colleagues (12, 13). In an extension of these efforts, we describe a one-dimensional analogue of the HMBC experiment ( 14)) for which we propose the acronym SIMBA (selective inverse multiple bond analysis). With this new experiment, each proton with a resolved long-range coupling to the selected carbon simultaneously appears in the resultant spectrum with an apparent

Improvements in the sensitivity of inverse-detected heteronuclear correlation spectra using micro inverse probes and micro cells: HMQC and HMBC spectra of caribbean ciguatoxin — preliminary structural inferences

Abstract Ciguatoxins are extremely potent activators of voltage-dependent sodium channels and elicit gastrointestinal and neurologic dysfunction in humans. Because they are present only at extremely low levels, the acquisition of heteronuclear shift correlation nmr spectra has been very difficult. Inverse-detected heteronuclear shift correlation experiments (HMQC and HMBC) have greatly increased sensitivity relative to older, heteronuclide-detected experiments. The use of micro inverse-detection probes has afforded a further dramatic increase in sensitivity relative to conventional 5 mm probe technology. Micro inverse-detection probes employed in conjunction with Shigemi micro nmr cells are shown to afford a further significant improvement in sensitivity which was necessary to acquire spectra on a sample of

Brevetoxin-3: Total assignment of the 1H and 13C NMR spectra at the submicromole level

Abstract The proton and carbon NMR spectra of the marine polyether toxin, brevetoxin-3, are totally assigned using a series of 2D NMR experiments which included: TOCSY, ROESY, HMQC, HMBC, and IDR-(Inverted Direct Response)-HMQC-TOCSY. All work was performed on a sample consisting of 800 μg (0.95 μmole) at 500 MHz.

The confromational behaviour of the cardiac glycoside digoxin as indicated by NMR spectroscopy and molecular dynamics calculations

The 1H- and 13C-NMR spectra of digoxin in solution in Me2SO-d6 have been assigned completely. Measurement of the 3JC,H values has enabled estimation of the torsional angles involving the bonds linking the digitoxose residues, between the inner digitoxose and the genin unit, and for the unsaturated gamma-lactone ring. These values have been supplemented by 1H-1H NOE data. In general, there is good agreement between the conformations in solution (NMR data) and the solid state (X-ray data), and that derived from theoretical modelling which shows evidence of conformational flexibility. The major difference occurs for the torsion between the genin and the innermost digitoxose residue where molecular dynamics predict the presence of two conformations, one similar to that seen by NMR and the other similar to the X-ray structure.

1D HMQC-tocsy: A selective one-dimensional analogue of HMQ-TOCSY

Abstract A selective, soft-pulse one dimensional analogue of the HMQC-TOCSY experiment, 1D HMQC-TOCSY, is described. An application is presented for a carbocyclic adenosine analog wherein problems of overlap between vicinally coupled protons preclude assignment by traditional methods such as COSY or relayed COSY.

Metabolite structure elucidation using NMR micro-/nano-detection

The structure of the epoxide of 1-ethyl-phenoxathiin 10,10-dioxide, a potential monoamine oxidase-A inhibitor, was elucidated using a combination of 500 MHz homo- and heteronuclear NMR techniques. The proton reference spectrum and a COSY spectrum were obtained on a 30 μg sample (0.07 μMoles) of the metabolite in 35 μl of d6-DMSO using a Varian heteronuclear Nano-probe™. An inverse-detected (HMQC) heteronuclear shift correlation spectrum was obtained on the 30 μg sample of the metabolite in 120 μl of d6-DMSO using a Nalorac Z•SPEC™ 3 mm micro inverse probe.

Solution conformations of the 6α and 6β epimers of oxymorphamine

Abstract High field (360 MHz) 1 H NMR studies have shown that the conformation of ring C in the oxymorphamines is dramatically influenced by the stereochemistry of the 6-amino group: ring C exists in a chair conformation in the 6β-epimer but adopts a twist boat conformation in the 6α-epimer.

An iterative linear method for calculation of spin-lattice relaxation times

Abstract A simple algorithm for the calculation of spin-lattice relaxation times which can be run in a programmable calculator is presented. As was suggested by H. Hanssum et al. , an experimentally determined inhomogeneity parameter ( I ) can be used with this procedure to compensate for imperfections in the RF field. The effects of variation of pulse width and repetition rate on both I and T 1 are investigated with simulated and experimental data sets. The superiority of this approach over three-parameter nonlinear fitting methods is demonstrated by comparisons between data sets generated with different pulse flip angles and sample volumes.

Nuclear magnetic resonance and X-ray stereochemical studies on 1-benzyltetrahydroisoquinolinium salts

The relative stereochemistry at positions 1 and 2 of N,N-dialkyl-1-benzyl-1,2,3,4-tetrahydroisoquinolinium salts has been assigned via a combination of 1H and 13C n.m.r. studies and confirmed by single-crystal X-ray analysis of trans-N-3-hydroxypropyl-5′-methoxylaudanosinium perchlorate [trans-2j)] and cis-N-3-hydroxypropyl-5′-methoxylaudanosinium iodide [cis-(2h)] monohydrate. Quaternization of either laudanosine or 5′-methoxylaudanosine with an alkyl iodide leads to major isomers which possess a trans-relationship between the entering N-alkyl group and the 1-benzyl substituent; these isomers characteristically have the less shielded 1-H and N-methyl protons in deuteriochloroform. Crystals of trans-(2j) and cis-(2h) monohydrate both belong to the monoclinic system, Space group P21/c, with a= 8.123(4), b= 20.380(8), c= 16.710(6)A, β= 102.14(5)°, Z= 4 for the former, and a= 11.475(5), b= 17.749(6), c= 14.278(5)A, β= 113.51(5)°, Z= 4, for the latter compounds. Full-matrix least-squares refinement of atomic positional and thermal parameters converged at R= 0.082 [2 215 reflections] for (trans-(2j) and 0.077 [2 987 reflections] for (cis-(2h) monohydrate. The stereochemistry of the protonation products arising from these tetrahydroisoquinoline bases is also discussed.