Swartz Analytical Techniques in Combinatorial Chemistry

NMR Methods

99

Figure 19 On-flow 500 MHz HPLC-NMR contour plot spectrum of chemical shift vs. retention time for a mixture of isomolecular weight dimethoxybenzoylglycines.

The simultaneous determination of structure by the combination of HPLC-NMR-MS should prove to be an extremely powerful tool for combinatorial chemistry.

B. Flow NMR

The ability to obtain routine NMR data in a reasonable time frame is greatly enhanced by the use of robotic sample changers. Currently it is possible to obtain data from over 100 samples in an unattended manner. The inefficiency in this procedure is that the samples generally are made by manual methods restricting the potential throughput and capacity. Mass spectrometry techniques have been developed to obtain spectra in about a minute or so each, using an autosampler that can take samples directly from 96 welled plates. These same plates are also used in the high-throughput screening efforts, thereby minimizing the effort to prepare samples. It would be convenient if

100

Shapiro

Figure 20 2D TOCSY spectrum for FNKEF-OH pentapeptide derivative obtained using stop-flow HPLC-NMR. Amino acid assignments are indicated by the letter code.

the same format could be used to obtain NMR spectra. Flow NMR has been recently introduced using a similar format MS and is commercially available. NMR most likely won’t be able to obtain data in the same time scale as MS; however, high-quality spectra can most probably be obtained in around 3 min. The robotics used to obtain these spectra will allow samples to be spot-checked or to permit one to obtain data on the identified active well as suggested in the cartoon in Fig. 21.

Since the solvent used in these wells is generally DMSO/water, this makes the flow NMR experiment that much more difficult. Techniques have been described to efficiently suppress the protons from multiple solvents and obtain NMR data on small samples in a relatively short time (57,58). Highquality NMR data on a 26- g sample can be obtained in just over 2 min using

NMR Methods

101

Figure 21 Schematic representation of flow NMR using 96 welled plates.

either a microprobe or an HPLC-NMR probe. The HPLC probe in principle can be adapted to handle streams of samples using a modified stop-flow procedure. The perfection of this technique will replace the automation systems as we presently know them and may render NMR tubes superfluous.

One aspect of combinatorial chemistry is the synthesis of mixtures of structurally related compounds. This facilitates a high throughput in both synthesis and screening. The split-and-mix synthesis technique produces a mixture of compounds as the final product (59,60). In developing the synthesis of large libraries, smaller test systems are studied to optimize synthetic strategies. The utility of NMR for studying intact mixtures has not been extensively demonstrated. 2D NMR methods such as TOCSY have been used in relatively simple mixture analysis (61). However, for compounds that have their spin systems insulated by groups such as esters or ethers, TOCSY methodologies are not sufficient for complete analysis.

102

Shapiro

Recently, the use of pulsed-field gradient (PFG) technology to obtain diffusion coefficients of molecules has been demonstrated as a useful technique for mixture analysis (53). Unlike any other 2D experiment, size-resolved or diffusion-resolved NMR assigns the resonances based on the diffusion coefficient for each proton (or other spin) in the molecule and therefore can be used to distinguish resonances arising from different molecules (63–70) (Fig. 22). A method that involves the use of PFG and TOCSY, called diffusionencoded spectroscopy (DECODES), simplifies mixture analysis by NMR (71). The combination of PFG and TOCSY ‘‘decodes’’ the spin systems, allowing individual components in complicated mixtures to be assigned. A typical DECODES spectrum obtained in this manner is shown in Fig. 23. The use of TOCSY aids the calculation of the diffusion coefficient and determination of molecular identity.

The synthesis and screening of mixtures of compounds offers increased efficiency and throughput compared to making and testing individual compounds. However, utilization of mixtures of compounds requires a method to determine which molecule in the mixture is responsible for the desired effect. Typically, mixtures of compounds are prepared by design and these mixtures are tested without separation. When there is evidence of sufficient activity,

Figure 22 Schematic representation of molecular translation in solution. The DOSY experiment spatially encodes the molecule in 1 and then DECODES the movement in 2. The spectrum indicates the translational movement by a decreased observed signal intensity.

NMR Methods

103

Figure 23 Selected region of TOCSY spectrum obtained via DECODES sequence of an ester mixture in benzene. Peaks arise from propyl acetate, ethyl butyryl acetate, and butyl levulinate.

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-

104

Shapiro

Figure 24 (a) 1D 400 MHz 1 H NMR spectrum of the nine-component mixture in CDCl3. (1) DL-isocitric lactone, (2) (S)-( )-O-acetylmandelic acid, (3) DL-N-acetylho- mocysteine thiolactone, (4) ( )-sec-butyl acetate, (5) propyl acetate; (6) isopropyl butyrate, (7) ethyl butyryl acetate, (8) butyl levulinate, (9) hydroquinine-9-phenanthryl ether. (b) PFG 1D 1 H NMR spectrum of the mixture without hydroquinine-9-phe- nanthryl ether. (c) PFG 1D 1 H 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).

NMR Methods

105

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 1H 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 1H 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 1H 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.

VII. FUTURE

‘‘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

106

Shapiro

REFERENCES

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– 141, 1996.

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 ν -HPLC/fast atom bom-

bardment 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.

10.E Geralt, J Rizo, E Pedroso. Application of gel-phase 13C-NMR to solid-phase peptide Synthesis. Tetrahedron 40:4141–4152, 1984.

11.D Liebfritz, W Mayr, R Oekonomopulos, J Jung. 13C NMR spectroscopic studies on the conformation during stepwise synthesis of peptides bound to solubilizing polymer supports. Tetrahedron 34:2045-2050 1978.

12.SL Mannatt, D Horowitz, R Horowitz, RP Pinnell. Solvent swelling for enhancement of carbon-13 nuclear magnetic resonance spectral information from insoluble polystyrenes. Anal Chem 52:1529–1534, 1980.

13.AJ Jones, CC Leznoff, PI Svirskaya. Characterization of organic substrates bound to cross-linked polystyrenes by 13C NMR spectroscopy. Org Magn Reson 18:236–240, 1982.

14.BE C Lossey, RG Cannon, WT Ford, M Periyasamy, S Mohanraj. Synthesis, reactions, and 13C FT NMR spectroscopy of polymer-bound steroids. J Org Chem 55:4664–4668, 1990.

NMR Methods

107

15.MM Azure, B Calas, A Cave, J Parello. Synthese peptidique en phase solide. Caracterisation par RMN du proton d’un peptide immobilise sur un support polyacrylique. C R Acad Sc Paris 303:553–556, 1986.

16.GC Look, CP Holmes, JP Chinn, MA Gallop. Methods for combinatorial organic synthesis: the use of fast 13C NMR analysis for gel phase reaction monitoring. J Org Chem 59:7588–7590, 1994.

17.GC Look, MM Murphy, DA Campbell, MA Gallop. Trimethylorthoformate: a mild and effective dehydrating reagent for solution and solid phase imine formation. Tetrahedron Lett 36:2937–2940, 1995.

18.SL Manatt, SF Amsden, CA Bettison, WT Frazer, JT Gudman, BE Lenk, JF Lubetich, EA McNelly, SC Smith, DJ Templeton, RP Pinnell. A fluorine-19 NMR approach for studying Merrifield solid-phase peptide synthesis. Tetrahedron Lett 21:1397–1400, 1980.

19.MJ Shapiro, G Kumaravel, RC Petter, R Beveridge. 19F NMR monitoring of a SNAr reaction on solid support. Tetrahedron Lett 37:4671–4674, 1996.

20.A Svensson, T Fex, J Kihlberg. Use of 19F NMR spectroscopy to evaluate reactions in solid phase organic synthesis. Tetrahedron Lett 37:7649–7652, 1996.

21.F Bardella, R Eritja, E Pedroso, E Geralt. Gel-phase 31P-NMR. A new analytical tool to evaluate solid phase oligonucleotide synthesis. Bioorg Med Chem Lett 3:2793–2796, 1993.

22.CR Johnson, B Zhang. Solid phase synthesis of alkenes using the Horner-Wad- sworth-Emmons reaction and monitoring by gel phase 31P NMR. Tetrahedron Lett 36:9253–9256, 1995.

23.Z Tian, C Gu, RRW Roeske, M Zhou, RL Van Etten. Synthesis of phosphotyro- sine-containing peptides by the solid-phase method. Int J Peptide Protein Res 42:155–158, 1993.

24.B Schneider, D Doskocilova, JJ Dybal. Motional restrictions and chain conformation in various swollen crosslinked polystyrene gels from 1H NMR line-shape analysis. J Polymer 26:253–259, 1985.

25.WL Fitch, G Detre, CP Holmes, JN Schoolery, P Keifer. High resolutions 1H NMR in solid phase organic synthesis. J Org Chem 59:7955–7956, 1994.

26.RC Anderson, MA Jarema, MJ Shapiro, JP Stokes, M Ziliox. Analytical techniques in combinatorial chemistry: MAS CH correlation in solvent-swollen resin. J Org Chem 60:2560–2651, 1995.

27.PA Keifer, L Baltosis, DM Rice, AA Tymiak and JN Shoolery. A comparison of NMR spectra obtained for solid-phase-synthesis resins using conventional highresolution, magic-angle-spinning, and high-resolution magic-angle-spinning probes. J Magn Reson A 119:65–75, 1996.

28.P Keifer. Influence of resin structure, tether length, and solvent upon the highresolution 1H NMR spectra of solid-phase-synthesis resins. J Org Chem 61: 1558–1559, 1996.

29.IE Pop, CF Dhalluin, BP Deprez, PC Melnyk, GM Lippens, AL Tartar. Monitoring of a three-step solid phase synthesis involving a heck reaction using magic angle spinning NMR spectroscopy. Tetrahedron 52: 12209–12222, 1996.

108

Shapiro

30.MJ Shapiro, unpublished work.

31.EL Hahn, Spin echoes Phys Rev 80:580 1950.

32.DL Rabinstein, KK Mills, EJ Strauss. Proton NMR spectroscopy of human blood plasma and red blood cells. Anal Chem 60:1380A–1391A, 1988.

33.T Wehler, J Westman. Magic angle spinning NMR: a valuable tool for monitoring the progress of reactions in solid phase synthesis. Tetrahedron Lett 37:4771– 4774, 1996.

34.HDH Stover, JM Frechet. Direct polarization 13C and 1H magic angle spinning NMR in the characterization of solvent-swollen gels. J Macromolecules 22: 1574–1576, 1989.

35.HDH Stover, JM Frechet. NMR characterization of cross-linked polystyrene gels. J Macromolecules 24:883–888, 1991.

36.E Giralt, F Alberico, F Bardella, R Eritja, M Feliz, E Pedroso, M Pons, J Rizo. Gel-phase NMR spectroscopy as a useful tool in solid phase synthesis. Innov Persp Solid Phase Synthesis Collect. Pap., Int Symp. R. Epton (ed.) 111–120, 1990.

37.RC Anderson, MJ Shapiro, JP Stokes. Structure determination in combinatorial chemistry: utilization of magic angle spinning HMQC and TOCSY NMR spectra in the structure determination of Wang-bound lysine. Tetrahedron Lett 36:5311– 5314, 1995.

38.IE Pop, CF Dhalluin, BP Deprez, PC Melnyk, GM Lippens, AL Tartar Monitoring of a three-step solid phase synthesis involving a heck reaction using magic angle spinning NMR spectroscopy. Tetrahedron 52:12209–12222, 1996.

39.A Kumar, RR Ernst, K Wuthrich. A two-dimensional nuclear overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross-relaxation networks in biological macromolecules. Biochem Biophys Res Commun 95:1–6, 1980.

40.WP Aue, E Bartholdi, RR Ernst. Two-dimensional spectroscopy. Application to nuclear magnetic Resonance. J Chem Phys 64:2229–2246, 1976.

41.MJ Shapiro, J Chin, RE Marti, MA Jarosinski. Enhanced resolution in MAS NMR for combinatorial chemistry. Tetrahedron Lett 38:1333–1336, 1997.

42.M Patek, M Lehl. Safety-catch anchoring linkage for synthesis of peptide amides by Boc/Fmoc Strategy. Tetrahedron Lett 32:3891–3894, 1991.

43.A Meissner, P Bloch, E Humpfer, M Spraul, OW Sorensen. Reduction of inhomogeneous line broadening in two dimensional high-resolution MAS NMR spectra of molecules attached to swelled resins in solid-phase synthesis. J Am Chem Soc 119:1787–1788, 1997.

44.K Nagayama, K Wuthrich, RR Ernst. two-dimensional spin echo correlated spectroscopy (SECSY) for 1H NMR studies of biological macrcomolecules. Biochem Biophys Res Commun 90:305–311, 1979.

45.Y Kim, JH Prestegard. Measurement of vicinal couplings from cross peaks in COSY spectra, J Magn Reson 84:9–13, 1989.

46.WE Mass, FH Laukien and DG Cory. Gradient, high resolution, magic angle sample spinning NMR. J Am Chem Soc 118:13085–13086, 1996.