Download (direct link):
the mixture is deconvoluted to identify the active component. Several approaches to identify interesting components in a mixture have been described (72-77). Methods that identify active components of mixtures without the need for deconvolution could eliminate ‘‘false positives’’ and greatly reduce the effort required to analyze mixtures. One such method under investigation is affinity MS (78-80).
A somewhat similar method, termed affinity NMR, has shown that the diffusion coefficient of a small molecule binding with a ‘‘receptor’’ in solution is significantly different from the small compound alone (81). Thus the inter-
Figure 24 (a) 1D 400 MHz 'H NMR spectrum of the nine-component mixture in CDCl3. (1) DL-isocitric lactone, (2) (S)-(+)-0-acetylmandelic acid, (3) DL-A/-acetylho-mocysteine thiolactone, (4) ( + )-sec-butyl acetate, (5) propyl acetate; (6) isopropyl bu-tyrate, (7) ethyl butyryl acetate, (8) butyl levulinate, (9) hydroquinine-9-phenanthryl ether. (b) PFG 1D 1H NMR spectrum of the mixture without hydroquinine-9-phe-nanthryl ether. (c) PFG 1D 1H NMR spectrum of the nine component mixture using LED sequence. Chemical shifts arising from compounds 1 and 2 are shown. All other shifts are from compound 9. The PFG conditions were the same as in (b).
acting molecules can be distinguished from inert molecules in a manner reminiscent of physical separation of molecules by affinity chromatography. Affinity NMR, using hydroquinine 9-phenanthryl ether as a model receptor, was successfully applied to a nine-component mixture containing seven inert materials and the two carboxylic acids shown.
Figure 24 shows the normal 1D 'H NMR spectrum for the nine component mixture; without PFG, a control experiment performed on the mixture in the absence of hydroquinine 9-phenanthryl ether under identical PFG conditions and the 1D 'H NMR spectrum of the same mixture under the PFG conditions. Only signals from hydoquinine 9-phenanthryl ether and compounds 1 and 2 are observed in the bottom spectrum. As expected no NMR signals are present in the absence of molecular interactions. The structures of the compounds that interacted with hydroquinine 9-phenanthryl ether were identified directly in the mixture using the DECODES method.
Since the relatively high concentration of each component required by NMR adds up to a high total concentration of compounds for the mixture, the application of this methodology to screen combinatorial chemistry mixtures for biological activity will likely be limited by the total compound concentration tolerated by the biological target. Nevertheless, this NMR method, when applied to suitable systems, should add a powerful tool for mixture analysis.
C. SAR by NMR
A method for identifying active compounds from a library of low molecular weight ligands using 15N-labeled proteins has been recently reported (82). The binding is determined by the 15N or 'H chemical shift changes in the protein upon the addition of the ligand. The method, which at present is limited to small biomolecular receptors, promises to play an integral part of the drug discovery process.
‘‘I am not aware of any other field of science outside of NMR that offers so much creative freedom and opportunity for a creative mind to invent and explore new experimental schemes that can be fruitfully applied in a variety of disciplines.’’—Richard Ernst, Nobel Prize, 1993
1. CA Fyfe. Solid State NMR for Chemists; CFC Press: Guelph, Ontario, 1983
2. B Yan, G Kumaravel, H Anjaria, A Wu, R Petter, C Jewell, J Wareing. Infrared spectrum of a single resin bead for real-time monitoring of solid-phase reactions. J Org Chem 60:5736-5738, 1995.
3. B Yan, G Kumaravel. Probing solid-phase reactions by monitoring the IR bands of compounds on a single ‘‘flattened’’ resin bead. Tetrahedron 52:843-848, 1996.
4. JA Boutin, P Hennig, PS Bertin, L Petit, J-P Mahieu, B Serkiz, J-P Volland, J-L Fauchere. Combinatorial peptide libraries: robotic synthesis and analysis by nuclear magnetic resonance, mass spectrometry, tandem mass spectrometry, and high performance capillary electrophoresis techniques. Anal Biochem 234:126-
5. BJ Egner, GJ Langley, M. Bradley. Solid phase chemistry: direct monitoring by matrix-assisted laser desorption/ionization time of flight mass spectrometry. A Tool for Combinatorial Chemistry. J Org Chem 60:2652-2653, 1995.
6. K McMellop, W Davidson, G Hansen, D Freeman, N Pallai. The characterization of crude products from solid-phase peptide synthesis by v-HPLC/fast atom bombardment mass spectrometry. Peptide research 4:40-46, 1991.
7. H Sterlicht, GL Kenyon, EL Packer, J Sinclair. J Am Chem Soc 93:199-208, 1971.
8. R Epton, P Goddard, KJ Ivin. Gel phase 13C NMR spectroscopy as an analytical method in solid (gel) phase peptide synthesis. Polymer 21:1367-1371, 1980.
9. FGW Butwell, R Epton, EJ Mole, N Muzaffar, S Phillips. Deprotection studies in ultra-high load solid (gel) phase peptide synthesis. 13C NMR investigation of the efficacy of boron trifluoride-based side-chain deprotection cocktails. Innov Persp Solid Phase Synth Collect Pap Int Symp 121-132, 1990.