- Email: [email protected]
New methods for analyzing compounds on polymeric supports
94
New methods for analyzing compounds on polymeric supports Mark A Gallop* and William L Fitcht Beyond specialized applications in peptide and oligonucleotide synthesis, widespread utilization of solid-phase methods in organic chemistry has been hampered by a lack of powerful analytical methods for characterization of polymer-supported compounds. The advent of combinatorial organic synthesis has recently spawned efforts to develop spectroscopic techniques such as infrared spectrometry, mass spectrometry and nuclear magnetic resonance as routine tools for structure elucidation in solid-phase synthesis.
Addresses
Affymax Research Institute, 4001 Miranda Ave, Palo Alto, CA 94304, USA "e-mail: [email protected] ~e-mail: [email protected] Current Opinion in Chemical Biology 1997, 1:94-100
http://biomednet.com/elecref/136?593100100094 © Current Biology Ltd ISSN 1367-5931 Abbreviations evaporative light-scattering detection ELSD electrospray ionization mass spectrometry ESI-MS FTIR Fourier transform infrared spectroscopy gas chromatography GC high performance liquid chromatography HPLC IR infrared spectroscopy
liquid chromatography matrix assisted laser deabsorption/ionization magic angle sample spinning mass spectroscopy nuclear magnetic resonance spectroscopy TLC thin layer chromatography TOF-SIMS time-of-flight secondary ion mass spectrometry LC
MALDI MAS MS NMR
Introduction Advances in synthetic organic chemistry have always gone hand-in-hand with advances in organic analytical chemistry. While pioneering chemists around the turn of the century were dependent on melting point measurement and combustion analysis for compound identification and characterization, the accelerated developments in synthesis from the 1950s through the present day have been possible because of the continued evolution of sophisticated spectroscopic methods. Solid-phase synthesis is at the core of the split/pool method of combinatorial chemistry, as well as numerous high-throughput parallel synthesis approaches that provide discrete compounds. Analytical methods specific to combinatorial synthesis have been discussed in reviews of solid-phase synthetic methodologies [1,2,3"], in more specialized publications concerned with analytical aspects of combinatorial chemistry [4,5] and in a review of polymer-supported catalysts [6].
Every, reaction needs to be 'followed' in some way. This process is critical for determining when the reaction is complete, for optimizing reaction yields, in applying standard procedures to new substrates, in reaction scale-up etc. In a typical solution phase reaction: components A and B react in the presence of reagents or catalysts to give the desired product(s) C and by-product(s) D. The chemist can follow this reaction by watching for a colour, phase or pH change, by following the disappearance of A or B or a reagent, or by monitoring the appearance of C or D. The favoured tools for tracking compound appearance or disappearance are thin layer chromatography (TLC) and (less frequently) gas chromatography (GC), liquid chromatography (LC), nuclear magnetic resonance spectroscopy (NMR), and mass spectrometry (MS). In solution-phase chemistry, one will start a reaction with weighed amounts of pure A and B. The weight of the purified product C then allows calculation of the reaction yield. This is a key parameter, t, seful for comparing different reactions or the applicability of a reaction to different substrates. Following reactions in the solid phase is more challenging. In a typical solid-phase reaction components A and C are covalently attached to the polymeric support (resin), making their appearance or disappearance more difficult to follow: Component B and reagents are typically present in large excess, so their concentrations do not change appreciably during the reaction. In solid-phase chemistry the additional issue of polymer loading must be considered. Loading is the measure of how much ligand or reactive functional group is associated with the resin per unit weight. The measurement of loading is necessarily gravimetric because resins arc insoluble, and is usually described in units of millimoles per gram (mmol g-l). The yield of a solid-phase reaction is given bv comparing the loading of the starting material with the loading of the product (it will often be reported as the weight of cleaved pure product, though this is formally the yield of the two step process, reaction and cleavage). Combustion elemental analysis, titrametric analysis or gravimetric analysis of intermediates are frequently reported as methods for measuring loading, in spite of difficulties caused by the 'dilution factor' of the solid support (typically the solid phase consists of 80-95% matrix plus linkers and only 5-20% of reacting ligand) and by the danger of entrained impurities. Characterization of reaction products using conventional solution-phase analytical methods after cleavage from the polymeric support is always an important analysis option. Care should be taken not to rely exclusively on HPLC
New methods for analyzing compounds on polymeric supports Gallop and Fitch
purity of cleaved products as a measure of yield for a multistep solid-phase sequence. This practice is common, dcspite the high variability of UV detector responses to different organic compounds. Significant efforts have been made over the last two years to employ common spectroscopic techniques (including IR, MS and NMR) for the analysis of organic compounds while still attached to resin particles. A major motivation behind these studies has been to provide more convenient methods for monitoring solid-phase synthesis and to enable optimized reaction conditions to be more rapidly defined. A summary of recent advances in the application of such methods in combinatorial chemistry form the remainder of this review.
Infrared spectroscopy Infrared spectroscopy has been favored for many years as a convenient and sensitive technique for characterizing polymer-supported organic compounds [7]. Monitoring either the appearance or disappearance of an IR chromophore in a molecule can provide a straightforward method for following the kinetics of solid-phase reaction. Recent developments in Fourier-transform IR (FTIR) technology are having an important impact on the application of this technique in combinatorial synthesis. F T I R microscopy can be used to generate excellent spectra from single resin beads placed directly on the NaCI sample window of the spectrophotometer [8,9",10], or contained in a KBr pellet [11]. The spectra obtained from individual beads are comparable to those obtained from bulk resin samples, and Yah and Kumaravel at Sandoz have shown that recording transmission spectra on flattened resin particles significantly enhances the spectral resolution by providing a more uniform pathlength for the IR irradiation [12]. Spectral enhancements have also been obtained by using photo-acoustic F T I R methods, where the IR radiation absorbed by an analyte is detected as an acoustic wave generated in an inert gas in contact with the sample. For example, photo-acoustic-FTIR (PA-FTIR) spectra of resin beads were found to be free of baseline artifacts caused by light scattering and sample inhomogeneity in typical F T I R experiments [13]. The sensitivity of F T I R microscopy in these single bead experiments is impressive: by using an attenuated total reflectance (ATR) microscope objective, it is possible to collect IR absorption data from just a fraction of a bead's surface. It has been estimated that an ester carbonyl absorption band can be detected from as little as 130 fmol of material (or -0.025% of the total bead loading) [9"]. This approach has been used to monitor the kinetics of esterification of hydroxyl groups on TentaGel ® and Wang resins, and to compare the acylation rates of surface hydroxyls with hydroxyl groups embedded within a bead. Interestingl>, Wang acylation proceeded more rapidly than the corresponding TentaGel ® reaction, but there was no significant difference between the reactivity of the external and internal residues. This suggests that diffusion
95
of substrate into these resins is not rate limiting for this reaction. Russell and co-workers [11] have demonstrated that IR absorbances from deuterated protecting groups can provide a robust and quantitative measurement of deuterium content in an immobilized substrate, and that this can be used to monitor reaction progress and yields with such molecules. The carbon-deuterium (C-D) stretching absorbancc falls in a largely interference-free spectral region (2300-2200 cm -1), and for appropriate groups (e.g. Boc-d 9) the molar absorptivity of the C - D stretch was found to be independent of the chemical environment of the deuterated moiety. Russell and co-workers [14 °] have also developed a specialized flow-through IR cell that permits real-time in situ IR analysis of solid-phase organic reactions [14"]. In contrast to the single-bead reaction monitoring studies described earlier, this experimental set-up permits online analysis of reaction kinetics and can provide unique insights into the rates of reagent diffusion and reactivity within the microenvironment of an individual bead. Mass spectrometry and chromatography Mass spectrometry (MS) techniques provide the most generally useful methods for characterizing combinatorial mixtures, and individual products of solid-phase synthesis. Since the pioneering MS studies of peptide library pools by Jung and Beck-Sickinger [15] there have been numerous reports demonstrating the application of MS to the analysis of small organic molecule libraries. Electrospray ionization MS (ESI-MS) has been used to evaluate pools of up to 55 xanthene derivatives formed by aminolysis of a diacid chloride with a diverse mixture of 10 amines [16,17 °] Both positive ion and negative ion detection methods were applied to the sample, and collectively the two analysis modes detected -80% of the expected molecular ions. The majority of the missing ions corresponded to products derived from a specific amine that reacted poorly in the acylation. As anticipated, positively charged analytes gave much stronger spectra when analyzed in the positive ion mode. One significant conclusion drawn from this study is that estimating the relative ratios of prodt,cts in a combinatorial mixture based solely on molecular ion intensities is not generally likely to be valid because of variations in compot, nd ionization efficiencies. In instances where molecular weight redundancy of pool members introduced ambiguity into tile analysis, tandem mass spectrometry (MS/MS) was applied to generate molecular fragments characteristic of a particular compound [17°]. Side products arising from incomplete couplings could also be identified in this experiment, suggesting that MS analysis of small pools should be useft,1 in optimizing reaction conditions for a set of building blocks..Mixtures of eight dihydroisoquinolines or tetrahydroisoqt, inolines generated in a solid-phasc Bischler-Napieralski synthesis have also aft)>rded ESI mass spectra containing the fidl set of expccted molecular ions [18].
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Combinatorialchemistry
Combining chromatographic sample separation (either LC or GC) with mass detection offers even more powerful approaches to characterizing pooled library samples than simple direct-infusion MS. From a diketopiperazine library containing 10 pools of 10 compounds per pool, LC-MS analysis identified 96 of the expected 100 products [19]. A pool of nine bicyclic proline analogs formed via a three-component cycloaddition reaction gave a complex LC-MS chromatogram due to the formation of multiple stereoisomers, though all the anticipated molecular ions were readily detectable [20]. High quality GC-MS spectra from mixtures of heteroarvl anilides and ethers have clearly established the utility of GC methods in characterizing modestly sized pools of volatile compounds [21,22]. Mass spectral techniques have also been successfully employed in determining the structures of small organic molecules present on single resin beads [23-26,27°,28°]. Brummel et al. [27"] have contrasted the utility of ESI, matrix-assisted laser desorption ionization (MALDI) and time-of-flight secondary ion MS (TOF-SIMS) in single bead MS studies. While each method provides adequate sensitivity for addressing single beads, the ESI and MALDI methods generate more abundant molecular ion signals and structurally informative fragment ions. T h e TOF-SIMS approach requires the most sophisticated experimental set-up, but provides the most accurate measurement of ion mass, and can be used to image samples in two-dimensional arrays [24]. Samples attached to resin via an acid-labile linkage are typically exposed to liquid or vapour T F A (trifluoroacetic acid) before MS analysis. In ESI-MS, the acid-treated bead is extracted with a small quantity of an appropriate solvent (e.g. MeCN, MeOH) and the sample introduced by flowinjection electrospray. In M A L D I experiments, the bead is first impregnated with a solution of dihydroxybenzoic acid (the activating matrix) and the sample irradiated with multiple pulses of a 3 3 7 n m N 2 laser. Researchers from Scripps have reported that compounds attached to resin via a photolabile ¢~-methyl-phenacyl ester linker can be directly analyzed by MALDI-MS without the need for prior cleavage of the covalent linkage [28"]. A single laser pulse directed at a Wang resin bead containing an undecapeptide sequence gave a clear molecular ion signal. This one-step MALDI procedure for direct analysis of resin-bound molecules has been used to monitor the extent of coupling of an activated amino acid residue to a peptide. It was noted that, in this case, a free amino group in the analyte (i.e. a site of protonation) appeared essential for generating a MALDI response. A remarkable feature of this experiment is that it required photolysis in the solid-phase followed by sample diffusion into the matrix, protonation and transfer to the gas phase. In our hands, a similar experiment required the additional expediency of adding solvent to the photocleaved bead in order to
promote diffusion and subsequent cocrystallization of the analyte and matrix. Laser irradiation of the bead then afforded an excellent M A L D I / T O F signal. Library strategies that use high speed parallel compound synthesis to generate arrays of discrete compounds (as opposed to mixtures generated via the split resin approach) have been assuming greater significance in the drug discovery communit'y: Obtaining detailed analytical data on such product arrays is generally accepted as an important component of library quality control, and has placed increased demands on analysis throughput and data management. Several groups engaged in large scale parallel synthesis programs have described the integration of robotic synthesis systems with MS or LC-MS autosampling interfaces that permit MS data to be acquired on some or all of the compounds prepared in a synthesis run [29°,30,31]. Samples for which unsatisfactory data are obtained (e.g. estimated product yield below some threshold level) can be flagged for further attention. One analytical technique that has recently emerged as a potentially useful method for estimating library synthesis yields is H P L C with evaporative light-scattering detection (ELSD) [32]. In ELSD, the H P L C column effluent is mixed with a high-velocity stream of nitrogen gas and nebulized. T h e mobile phase is rapidly evaporated in a heated tube and the non-volatile solute is swept through a laser beam causing light scattering, which is detected by a photodiode. ELSD is more universal than UV detection; in general it detects any analyte above a molecular weight of 200 atomic cnass units. Another new development is the chemiluminescent nitrogen detector (CLND) for H P L C [33]. This destructive detector (which oxidizes nitrogen compounds to NO, then converts the N O to excited NO 2 by reaction with ozone and detects the emitted light in a photomultiplier) has been shown to give equivalent response per mole of N for all nitrogen compounds tested to date. It remains to be seen how robust this detector is in regular practice, but it offers the tantalizing promise of quantitative organic analysis of nitrogen containing compounds without purified reference samples for each analyte.
N M R spectroscopy N M R is the most powerful tool routinely used in solutionphase organic synthesis for structural characterization of small organic molecules. There has consequently been significant interest in the application of NMR spectroscopic methods for characterizing products of solid-phase synthesis and for monitoring the progress of chemical transformations on resins. Because the line widths in NMR spectra are greatly affected by both the mobility of atoms in a sample as well as tile homogeneity of the sample matrix, compound immobilization on solid
New
methods
for analyzing
supports dramatically influences the type and quality of spectral information that can be obtained by NMR.
on
polymeric
supports
Gallop
and
Fitch
97
phase synthesis of building blocks selectively enriched with I.~C enables the gel-phase method to be employed for convenient rapid reaction monitoring. Spectra with excellent signal-to-noise ratios can be obtained from -20 mg of resin in a 5 mm N M R tube in just a few minutes. Although the commercial availability of labeled starting materials is somewhat limited, we are frequently able to use this fast 13C N M R method for optimizing solid-phase synthetic protocols.
Early workers studying solid-phase peptide and oligonucleotide chemistry described the application of gel-phase muhinuclear N M R spectroscopy to the characterization of polymer-supported products [34]. In gel-phase NMR, a spectrum is acquired using a conventional liquids probe from a polymer sample that has been swollen in a suitable solvent. In comparison with solution-phase spectra, the signals obtained by the gel-phase method are relatively broad, and useful information is only obtained for nuclei such as ]3C, 31p and 19F which have wide spectral dispersion (for recent examples of applications of gel-phase 31p and 19F NMR, see [35-37]). Moreover, with gel-phase carbon NMR the low abundance of the 13C nucleus demands prolonged spectral acquisition times and the sample's spectrum may frequently be confounded by signals resulting from the polymer matrix. Gallop and co-workers [38,39] have described how the use in solidFigure
compounds
While 1H N M R is sufficiently sensitive to permit rapid analysis of solid-phase reactions, the signals obtained from resin samples by the gel-phase approach are typically too broad (>>25 Hz) to be useful in structure determinations. This line broadening can result from the restricted molecular mobility of the tethered compound, as welt as from magnetic field inhomogeneity surrounding the sample (susceptibility mismatches at the resin/solvent interface) and homonuclear dipolar interactions. These latter effects can be minimized by rapidly spinning the
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The 300 MHz MAS 1H NMR spectrum of TentaGel ~ resin-bound I}-Iactams. Note that the product is a 3:1 mixture of racemic cis lactams, with only the ~-configuration shown for convenience. Complete assignment of the lactam resonances has been made through a combination of COSY and decoupling studies. Several groups using MAS have successfully applied conventional two-dimensional NMR experiments (e.g. TOCSY, NOESY, 13C-1H HMQC) to resin samples using MAS [41,43,47,51].
98
Combinatorial chemistry
sample at the 'magic angle', and a number of recent reports have described how high-resolution 1H NMR spectra can bc obtained from resin-bound compounds using the magic angle sample spinning (MAS) technique [40-13]. Optimal spectra have been obtained from samples spun at - 2 k H z using very low volume (<40 p.L) NlklR probes (the Varian Nano-NMR probe) having careful susceptibility matching, although conventional solid state CP/MAS probes have been found to give good quality 1H NMR spectra also [44"]. Keifer [45"1 has published a detailed comparison of IN MAS spectra for the same organic sample attached to a range of synthesis resins using a variety of different solvents. These studies indicate that the resin structure is the dominant factor influencing line widths, with the solvent playing a secondary role. TentaGel ® resins, which have long, mobile polyethylene glycol tethers, generally provide the best line shapes, with line widths o f - 4 Hz being observed in CDzCIz. Each different type of resin (e.g. Wang, Merrifield, TentaGel @, etc.) gives a characteristic set of background signals, including broad aromatic resonances arising from rotationallv restricted nuclei in the polystyrene core. Presaturation or the application of spin-echo pulse sequences [46] are effective means of reducing these unwanted interferences, and can provide ill NMR spectra that are virtually equivalent in quality to those generated from soluble samples. An example of this type of high-resolution 1H MAS NMR spectrt, m, obtained from an isomeric mixture of monocyclic ]3-1actams produced via ketene cycloaddition to a resin bound imine, is shown in Figure 1 [47]. While high-resolution lit MAS spectra are routinely acquired in minutes from just 5 mg of resin, the sensitivity of the Nano-NMR probe is such that interpretable information can even bc obtained from a single resin bead. A 1-~C-filtered IH spectrum of (3,5-dimethoxy-l-~C) benzoic acid attached to a single bead of Wang resin (-800 pmol sample) clearly shows the expected doublet signal for the methoxy protons around 3.Tppm, though isotope editing was required to eliminate interferences from contaminants and solvent [48]. However, the limited amount of information that can be obtained from this type of single bead NMR analysis effectively makes the experiment a novelty More useful data have recently been obtained from single beads of a macro TentaGel~-type resin (400-750~m diameter. 10-65nmol sample) where direct 114 observation in a CP/MAS probe gave clear assignable resonances, and enabled intermediates in the synthesis of a hydantoin moiety to be readily characterized [49"]. Lin and Shapiro [50] have explored the use of pt, lsed-tield gradient technology with the TOCSY experiment as a means of obtaining useful NMR from mixtures of several compounds (e.g. a small combinatorial compound pool). This method assigns resonances on the basis of the diffusion coefficient for each proton in a moleculc, and
thus can distinguish resonances arising from different compounds. It is unclear how complex such mixtures can become before this techniqne is impractical. Conclusions
The mainstream analytical methods for organic structure elucidation, familiar from solution-phase synthesis, are rapidly being co-opted by solid-phase chemists. As the use of combinatorial techniques expands in areas as diverse as pharmaceutical research and new materials discover~, we can anticipate a dramatic increase in the number of reports of new solid-phase synthetic methods appearing in the scientific literature. The arbiters of quality control in organic chemistry determine which analytical methods will be used for proving the structure and purity of new organic compounds, and these standards are upheld through the editorial policies of chemical journals. While there are, as yet, no generally accepted standards for characterization of polymer-supported products, it would seem that appropriate analytical tools are becoming sufficiently widespread in the research community that formal analytical criteria could soon be established.
References
and
recommended
reading
Papers of particular interest, published within the annual period of review, have been highlighted as: * .-
of special interest of outstanding interest
1.
Hermkens PHH, Ottenheijm HCJ, Rees D: Solid phase organic reactions: a review of the literature. Tetrahedron 1996, 52:4527-4554.
2.
Fruchtel JS, Jung G: Organic chemistry on solid supports. Angew Chem /nt Ed 1996, 35:17-42.
3. Thompson LA, EIIman JA: Synthesis and applications of small • molecule libraries. Chem Rev 1996, 96:555-600. A review of small molecule library synthesis. Includes a useful compendium of analytical methods for solid-phase chemistry. 4.
Fitch WL (Ed): Analytical Methods for Quafity Control of Combinatorial Libraries, vol 1. Leiden: ESCOM Science Publishers; 1997, in press.
5.
Fitch WL, Detre G, Look GC: Combinatorial Chemistry and Molecular Diversity in Drug Discovery. Edited by Kerwin JF, Gordon EM. New York: John Wiley & Sons; 1997, in press.
6.
Schlick S, Bortel E, Dyrek K: Catalysis on polymer supports. Acta Po/ym 1996, 47:1-15.
7.
Crowley JI, Rapoport H: Solid-phase synthesis: novelty or fundamental concept? Accounts Chem Res 1976, 9:135-144.
8.
Yah B, Kumaravel G, Anjara H, Wu A, Petter RC, Jewell CF Jr, Wareing JR: Infrared spectrum of a single resin bead for realtime monitoring of solid-phase reactions. J Org Chem 1995, 60:5736-5738.
9. •,
Yan B, Fell JB, Kumaravel G: Progression of organic reactions on resin supports monitored by single bead FTIR microspectroscopy. J Org Chem 1996, 61:7467-7472. Very nice full paper, describing the use of single bead FTIR in a microscope instrument. Individual beads can be imaged under a microscope objective to provide spectra that approach the quality of those recorded on bulk resin samples. FTIR microscopy has excellent sensitivity and can detect strong IR chromophores (e.g. ester carbonyls) at the sub-picomole level. This method can also be useful for following the kinetics of polymer-supported reactions. 10.
Yan B, Gstach H: An indazole synthesis on solid support monitored by single bead FTIR microspectrocopy. Tetrahedron Lett 1996, 37:8325-8328.
New methods for analyzing compounds on polymeric supports Gallop and Fitch
11.
Russell K, Cole DC, McLaren FM, Pivonka DE: Analytical techniques for combinatorial chemistry: quantitative infrared spectroscopic measurements of deuterium-labeled protecting groups. J Am Chem Soc 1996. 118:7941-7945.
12.
Yan B, Kumaravel G: Probing solid-phase reactions by monitoring IR bands of compounds on a single 'flattened' resin bead. Tetrahedron 1996, 52:843-848.
13.
Gosselin F, Di Renzo M, Ellis TH, Lubell WD: Photoacoustic FTIR spectroscopy, a nondestructive method for sensitive analysis of solid-phase organic chemistry. J Org Chem 1996, 61:7980-7981.
14, •
Pivonski DE, Russell K, Gero T: Tools for combinatorial chemistry: in situ infrared analysis of solid-phase organic reactions. Appl Spectrosc 1996, 50:1471-1478. An impressive study describing a device for real time monitoring o1 solid phase reactions. Beads are trapped in an IR cell set up to allow reagents to flow through. FTIR spectra are then recorded and analyzed. Functional group changes can be monitored and optimized. 15.
Jung G, Beck-Sickinger AG: Multiple peptide synthesis methods and their applications, Angew Chem Int Ed 1992, 31:367-383.
16.
Carell T, Wintner EA, Sutherland AJ, Rebek J Jr, Dunayevskiy YM, Vouros P: New promise in combinatorial chemistry: synthesis, characterization, and screening of small-molecule libraries in solution. Chem Biol 1995, 2:1 ? 1-183.
17. •
Dunayevskiy YM, Vouros P, Carell T, Wintner EA, Rebek J Jr: Characterization of the complexity of small-molecule libraries by electrospray ionization mass spectrometry. Anal Chem 1995, 67:2906-2915. Study demonstrates utility of ESI-MS for analysis of soluble compound pools of moderate complexity.
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Discusses practical considerations in establishing an automated high throughput system for mass spectrometric analysis of thousands of combinatorial samples per month in a pharmaceutical company environment. 30.
Garr CD, Peterson JR, Schultz L Oliver AR, Underiner TL, Cramer RD, Ferguson AM, Lawless MS, Patterson DE: Solutionphase synthesis of chemical libraries for lead discovery. J Biomol Screening 1996, 1:179-186.
31.
Cargill JF, Maiefski RR: Automated combinatorial chemistry on solid phase. Lab Robotics Automat 1996, 8:139-148.
32.
Kibbey CE: Quantitation of combinatorial libraries of small organic molecules by normal-phase HPLC with evaporative light-scattering detection, Mol Divers 1995, 1:24?-258.
33.
Fitch WL, Szardenings AK, Fujinari EM: Chemiluminescent nitrogen detection for HPLC: an important new tool in organic analytical chemistry. Tetrahedron Lett 1997, 38:1689-1692.
34.
Giralt E, Albericio F, Bardella F, Eritja R, Feliz M, Pedroso E, Pens M, Rizo J: Gel-phase NMR spectroscopy as a useful tool in solid-phase synthesis. In Innovation and Perspectives in SolidPhase Synthesis. Edited by Epton R. Birmingham: SPCC (UK) Ltd; 1990:111-120.
35.
Johnson CR, Zhang B: Solid-phase synthesis of alkene using the Horner-Wadsworth-Emmons reaction and monitoring by gel-phase 31p NMR. Tetrahedron Lett 1995, 36:9253-9256.
36.
Shapiro M J, Kumaravel G, Petter RG, Beveridge R: lSF Monitoring of a SNAr reaction on solid support. Tetrahedron Lett 1996, 37:4671-46?4.
3?.
Svensson A, Fex T, Kihlberg J: Use of 19F NMR spectroscopy to evaluate reactions in solid-phase organic synthesis. Tetrahedron Lett 1 996, 37:7649-7652.
38.
Look GC, Holmes CP, Chinn JP, Gallop MA: Methods for combinatorial organic synthesis: the use of fast 13C NMR analysis for gel-phase reaction monitoring. J Org Chem 1994, 59:7588-7590.
18.
Meutermans WDF, Alewood PF: The solid-phase synthesis of dihydro- and tetrahydroisoquinolines. Tetrahedron Lett 1995, 36:7709-7712.
19.
Gordon DW, Steele J: Reductive alkylation on a solid phase: synthesis of a piperazinedione combinatorial library. Bioorg Med Chem Lett 1995, 5:47-50.
39.
Hamper BC, Dukesherer DR, South MS: Solid-phase synthesis of proline analogs via a three component 1,3-dipolar cyeloaddition. Tetrahedron Lett 1996, 37:3671-3674.
Gordon EM, Gallop MA, Patel DV: Strategy and tactics in combinatorial organic synthesis. Applications to drug discovery. Accounts Chem Res 1996, 29:144-154.
40.
Parlow JJ, Normansell JE: Discovery of a herbicidal lead using polymer-bound activated esters in generating a combinatorial library of amides and esters. Mo/Divers 1995, 1:266-269.
Fitch WL, Detre G, Holmes CP, Shoolery JN, Keifer PA: High resolution 1H NMR in solid-phase organic synthesis. J Org Chem 1994, 59:7955-7956.
41.
Anderson RC, Jarema MA, Shapiro MJ, Stokes JP, Ziliox M: Analytical techniques in combinatorial chemistry: MAS CH correlation in solvent-swollen resin. J Org Chem 1995, 60:2650-2651.
42.
Wehler T, Westman J: Magic angle spinning NMR: a versatile tool for monitoring the progress of solid phase synthesis. Tetrahedron Lett 1996, 37:4771-4774.
43.
Pop IE, Dhalluin CE Deprez BP, Melnyk PC, Lippens GM, Tartar AL: Monitoring of a three step solid phase synthesis reaction involving a Heck reaction using magic angle spinning NMR spectroscopy. Tetrahedron 1996, 52:12209-12222.
20.
21.
22.
Parlow JJ: The use of anionic exchange resins for the synthesis of combinatorial libraries containing aryl and heteroaryl ethers. Tetrahedron Lett 1996, 37:5257-5260.
23.
Zambias RA, Boulton DA, Griffin PR: Microchemical structural determination of a peptoid covalently bound to a polymeric bead by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, Tetrahedron Lett 1994, 35:4283-4286.
24.
Brummel CL, Lee INW, Zhou Y, Benkovic SJ, Winograd N: A mass spectrometric solution to the address problem of combinatorial libraries, Science 1994, 264:399-402.
25.
Brown BB, Wagner DS, Geysen HM: A single bead decode strategy using electrospray ionization mass spectrometry and a new photolabile linker. Mo/Divers 1995, 1:4-12.
26.
Egner BJ, Langley G J, Bradley M: Solid-phase chemistry: direct monitoring by matrix-assisted laser desorption/ionization timeof-flight mass spectrometry. A tool for combinatorial chemistry. J Org Chem 1995, 60:2652-2653.
27. •
Brummel CL, Vickerman JC, Carr SA, Hemling ME, Roberts GD, Johnson W, Weinstock J, Gaitanopoulos D, Benkovic SJ, Winograd N: Evaluation of mass spectrometric methods applicable to the direct analysis of non-peptide bead-bound combinatorial libraries. Anal Chem 1996, 68:237-242. A comparison of ESI-MS, MALDI/TOF-MS and TOF/SIMS for characterization of the material from a single bead of a combinatorial library. Fitzgerald MC, Harris K, Shevlin CG, Siuzdak G: Direct characterization of solid phase resin-bound molecules by mass spectrometry. Bioorg Med Chem Lett 1996, 6:979-982. Direct MALDI of a peptide ligand on Wang resin with a photolinker. MALDI provides both photoc}eavage and ionization.
44. •
Keifer PA, Baltusis L, Rice DM, Tymiak AA, Shoolery JN: A comparison of NMR spectra obtained for solid-phasesynthesis resins using conventional high-resolution, magicangle-spinning and high-resolution magic-angle-spinning probes. J Magn Res 1996, 119:65-75. A comparison of the Nano-NMR probe with CP/MAS and conventional probes for 1H and 130 spectra of solid-phase resins and tethered molecules. 45. ••
Keifer PA: Influence of resin structure, tether length, and solvent upon the high-resolution 1H NMR spectra of solid phase synthesis resins. J Org Chem 1996, 61:1558-1559. An interesting comparison of 1H MAS NMR data obtained for several protected aspartic acid residues attached to a variety of commercially available resins. The most important factors influencing proton line-widths are resin structure and the NMR solvent, with polyethyleneglycol-grafted resins like TentaGel ~';swollen in CD2CI 2 affording the best resolution. 46.
28. •
29. •
Hegy G, Gorlach E, Richmond R, Bitsch F: High throughput electrospray mass spectrometry of combinatorial chemistry racks with automated contamination surveillance and results reporting. Rapid Commun Mass Spectrom 1996, 10:1894-1900.
Garigipati RS, Adams B, Adams JL, Sarkar SK: Use of spin echo magic angle spinning 1H NMR in reaction monitoring in combinatorial organic synthesis. J Org Chem 1996, 61:2911-2914.
47
Ruhland B, Bhandari A, Gordon EM, Gallop MA: Solid-supported combinatorial synthesis of structurally diverse I~-Iactams. J Am Chem Soc 1996, 118:253-254.
48.
Sarkar SK, Garigipafi RS, Adams JL, Keller PA: An NMR method to identify nondestructively chemical compounds bound to a
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single solid-phase-synthesis bead for combinatorial chemistry applications. J Am Chem Soc 1996, 118:2305-2306. 49. •
Pursch M, Schlotterbeck G, Tseng L, Albert K, Rapp W: Monitoring the reaction progress in combinatorial chemistry: 1H MAS NMR investigations on single macro beads in the suspended state. Angew Chem Int Ed 1996, 35:2867-2869. Using single beads of a macroscopic TentaGel ~ resin ( 4 0 0 - 7 5 0 p m diameter) containing 10-65 nmole of compound per bead, these workers were able to monitor the reaction sequence leading to hydantoin formation. Individual beads were placed in a four mm rotor of a CP/MAS probe and high
resolution proton NMR spectra were obtained with 128-160 scans in 6 - 8 minutes, demonstrating that single bead NMR can be practical for large resin particles. 50.
Lin M, Shapiro M J: Mixture analysis in combinatorial chemistry. Application of diffusion-resolved NMR spectroscopy. J Qrg Chem 1996, 61:761 7-7619.
51.
Anderson RC, Stokes JP, Shapiro M J: 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 1995, 36:5311-5314.
New methods for analyzing compounds on polymeric supports Mark A Gallop* and William L Fitcht Beyond specialized applications in peptide and oligonucleotide synthesis, widespread utilization of solid-phase methods in organic chemistry has been hampered by a lack of powerful analytical methods for characterization of polymer-supported compounds. The advent of combinatorial organic synthesis has recently spawned efforts to develop spectroscopic techniques such as infrared spectrometry, mass spectrometry and nuclear magnetic resonance as routine tools for structure elucidation in solid-phase synthesis.
Addresses
Affymax Research Institute, 4001 Miranda Ave, Palo Alto, CA 94304, USA "e-mail: [email protected] ~e-mail: [email protected] Current Opinion in Chemical Biology 1997, 1:94-100
http://biomednet.com/elecref/136?593100100094 © Current Biology Ltd ISSN 1367-5931 Abbreviations evaporative light-scattering detection ELSD electrospray ionization mass spectrometry ESI-MS FTIR Fourier transform infrared spectroscopy gas chromatography GC high performance liquid chromatography HPLC IR infrared spectroscopy
liquid chromatography matrix assisted laser deabsorption/ionization magic angle sample spinning mass spectroscopy nuclear magnetic resonance spectroscopy TLC thin layer chromatography TOF-SIMS time-of-flight secondary ion mass spectrometry LC
MALDI MAS MS NMR
Introduction Advances in synthetic organic chemistry have always gone hand-in-hand with advances in organic analytical chemistry. While pioneering chemists around the turn of the century were dependent on melting point measurement and combustion analysis for compound identification and characterization, the accelerated developments in synthesis from the 1950s through the present day have been possible because of the continued evolution of sophisticated spectroscopic methods. Solid-phase synthesis is at the core of the split/pool method of combinatorial chemistry, as well as numerous high-throughput parallel synthesis approaches that provide discrete compounds. Analytical methods specific to combinatorial synthesis have been discussed in reviews of solid-phase synthetic methodologies [1,2,3"], in more specialized publications concerned with analytical aspects of combinatorial chemistry [4,5] and in a review of polymer-supported catalysts [6].
Every, reaction needs to be 'followed' in some way. This process is critical for determining when the reaction is complete, for optimizing reaction yields, in applying standard procedures to new substrates, in reaction scale-up etc. In a typical solution phase reaction: components A and B react in the presence of reagents or catalysts to give the desired product(s) C and by-product(s) D. The chemist can follow this reaction by watching for a colour, phase or pH change, by following the disappearance of A or B or a reagent, or by monitoring the appearance of C or D. The favoured tools for tracking compound appearance or disappearance are thin layer chromatography (TLC) and (less frequently) gas chromatography (GC), liquid chromatography (LC), nuclear magnetic resonance spectroscopy (NMR), and mass spectrometry (MS). In solution-phase chemistry, one will start a reaction with weighed amounts of pure A and B. The weight of the purified product C then allows calculation of the reaction yield. This is a key parameter, t, seful for comparing different reactions or the applicability of a reaction to different substrates. Following reactions in the solid phase is more challenging. In a typical solid-phase reaction components A and C are covalently attached to the polymeric support (resin), making their appearance or disappearance more difficult to follow: Component B and reagents are typically present in large excess, so their concentrations do not change appreciably during the reaction. In solid-phase chemistry the additional issue of polymer loading must be considered. Loading is the measure of how much ligand or reactive functional group is associated with the resin per unit weight. The measurement of loading is necessarily gravimetric because resins arc insoluble, and is usually described in units of millimoles per gram (mmol g-l). The yield of a solid-phase reaction is given bv comparing the loading of the starting material with the loading of the product (it will often be reported as the weight of cleaved pure product, though this is formally the yield of the two step process, reaction and cleavage). Combustion elemental analysis, titrametric analysis or gravimetric analysis of intermediates are frequently reported as methods for measuring loading, in spite of difficulties caused by the 'dilution factor' of the solid support (typically the solid phase consists of 80-95% matrix plus linkers and only 5-20% of reacting ligand) and by the danger of entrained impurities. Characterization of reaction products using conventional solution-phase analytical methods after cleavage from the polymeric support is always an important analysis option. Care should be taken not to rely exclusively on HPLC
New methods for analyzing compounds on polymeric supports Gallop and Fitch
purity of cleaved products as a measure of yield for a multistep solid-phase sequence. This practice is common, dcspite the high variability of UV detector responses to different organic compounds. Significant efforts have been made over the last two years to employ common spectroscopic techniques (including IR, MS and NMR) for the analysis of organic compounds while still attached to resin particles. A major motivation behind these studies has been to provide more convenient methods for monitoring solid-phase synthesis and to enable optimized reaction conditions to be more rapidly defined. A summary of recent advances in the application of such methods in combinatorial chemistry form the remainder of this review.
Infrared spectroscopy Infrared spectroscopy has been favored for many years as a convenient and sensitive technique for characterizing polymer-supported organic compounds [7]. Monitoring either the appearance or disappearance of an IR chromophore in a molecule can provide a straightforward method for following the kinetics of solid-phase reaction. Recent developments in Fourier-transform IR (FTIR) technology are having an important impact on the application of this technique in combinatorial synthesis. F T I R microscopy can be used to generate excellent spectra from single resin beads placed directly on the NaCI sample window of the spectrophotometer [8,9",10], or contained in a KBr pellet [11]. The spectra obtained from individual beads are comparable to those obtained from bulk resin samples, and Yah and Kumaravel at Sandoz have shown that recording transmission spectra on flattened resin particles significantly enhances the spectral resolution by providing a more uniform pathlength for the IR irradiation [12]. Spectral enhancements have also been obtained by using photo-acoustic F T I R methods, where the IR radiation absorbed by an analyte is detected as an acoustic wave generated in an inert gas in contact with the sample. For example, photo-acoustic-FTIR (PA-FTIR) spectra of resin beads were found to be free of baseline artifacts caused by light scattering and sample inhomogeneity in typical F T I R experiments [13]. The sensitivity of F T I R microscopy in these single bead experiments is impressive: by using an attenuated total reflectance (ATR) microscope objective, it is possible to collect IR absorption data from just a fraction of a bead's surface. It has been estimated that an ester carbonyl absorption band can be detected from as little as 130 fmol of material (or -0.025% of the total bead loading) [9"]. This approach has been used to monitor the kinetics of esterification of hydroxyl groups on TentaGel ® and Wang resins, and to compare the acylation rates of surface hydroxyls with hydroxyl groups embedded within a bead. Interestingl>, Wang acylation proceeded more rapidly than the corresponding TentaGel ® reaction, but there was no significant difference between the reactivity of the external and internal residues. This suggests that diffusion
95
of substrate into these resins is not rate limiting for this reaction. Russell and co-workers [11] have demonstrated that IR absorbances from deuterated protecting groups can provide a robust and quantitative measurement of deuterium content in an immobilized substrate, and that this can be used to monitor reaction progress and yields with such molecules. The carbon-deuterium (C-D) stretching absorbancc falls in a largely interference-free spectral region (2300-2200 cm -1), and for appropriate groups (e.g. Boc-d 9) the molar absorptivity of the C - D stretch was found to be independent of the chemical environment of the deuterated moiety. Russell and co-workers [14 °] have also developed a specialized flow-through IR cell that permits real-time in situ IR analysis of solid-phase organic reactions [14"]. In contrast to the single-bead reaction monitoring studies described earlier, this experimental set-up permits online analysis of reaction kinetics and can provide unique insights into the rates of reagent diffusion and reactivity within the microenvironment of an individual bead. Mass spectrometry and chromatography Mass spectrometry (MS) techniques provide the most generally useful methods for characterizing combinatorial mixtures, and individual products of solid-phase synthesis. Since the pioneering MS studies of peptide library pools by Jung and Beck-Sickinger [15] there have been numerous reports demonstrating the application of MS to the analysis of small organic molecule libraries. Electrospray ionization MS (ESI-MS) has been used to evaluate pools of up to 55 xanthene derivatives formed by aminolysis of a diacid chloride with a diverse mixture of 10 amines [16,17 °] Both positive ion and negative ion detection methods were applied to the sample, and collectively the two analysis modes detected -80% of the expected molecular ions. The majority of the missing ions corresponded to products derived from a specific amine that reacted poorly in the acylation. As anticipated, positively charged analytes gave much stronger spectra when analyzed in the positive ion mode. One significant conclusion drawn from this study is that estimating the relative ratios of prodt,cts in a combinatorial mixture based solely on molecular ion intensities is not generally likely to be valid because of variations in compot, nd ionization efficiencies. In instances where molecular weight redundancy of pool members introduced ambiguity into tile analysis, tandem mass spectrometry (MS/MS) was applied to generate molecular fragments characteristic of a particular compound [17°]. Side products arising from incomplete couplings could also be identified in this experiment, suggesting that MS analysis of small pools should be useft,1 in optimizing reaction conditions for a set of building blocks..Mixtures of eight dihydroisoquinolines or tetrahydroisoqt, inolines generated in a solid-phasc Bischler-Napieralski synthesis have also aft)>rded ESI mass spectra containing the fidl set of expccted molecular ions [18].
96
Combinatorialchemistry
Combining chromatographic sample separation (either LC or GC) with mass detection offers even more powerful approaches to characterizing pooled library samples than simple direct-infusion MS. From a diketopiperazine library containing 10 pools of 10 compounds per pool, LC-MS analysis identified 96 of the expected 100 products [19]. A pool of nine bicyclic proline analogs formed via a three-component cycloaddition reaction gave a complex LC-MS chromatogram due to the formation of multiple stereoisomers, though all the anticipated molecular ions were readily detectable [20]. High quality GC-MS spectra from mixtures of heteroarvl anilides and ethers have clearly established the utility of GC methods in characterizing modestly sized pools of volatile compounds [21,22]. Mass spectral techniques have also been successfully employed in determining the structures of small organic molecules present on single resin beads [23-26,27°,28°]. Brummel et al. [27"] have contrasted the utility of ESI, matrix-assisted laser desorption ionization (MALDI) and time-of-flight secondary ion MS (TOF-SIMS) in single bead MS studies. While each method provides adequate sensitivity for addressing single beads, the ESI and MALDI methods generate more abundant molecular ion signals and structurally informative fragment ions. T h e TOF-SIMS approach requires the most sophisticated experimental set-up, but provides the most accurate measurement of ion mass, and can be used to image samples in two-dimensional arrays [24]. Samples attached to resin via an acid-labile linkage are typically exposed to liquid or vapour T F A (trifluoroacetic acid) before MS analysis. In ESI-MS, the acid-treated bead is extracted with a small quantity of an appropriate solvent (e.g. MeCN, MeOH) and the sample introduced by flowinjection electrospray. In M A L D I experiments, the bead is first impregnated with a solution of dihydroxybenzoic acid (the activating matrix) and the sample irradiated with multiple pulses of a 3 3 7 n m N 2 laser. Researchers from Scripps have reported that compounds attached to resin via a photolabile ¢~-methyl-phenacyl ester linker can be directly analyzed by MALDI-MS without the need for prior cleavage of the covalent linkage [28"]. A single laser pulse directed at a Wang resin bead containing an undecapeptide sequence gave a clear molecular ion signal. This one-step MALDI procedure for direct analysis of resin-bound molecules has been used to monitor the extent of coupling of an activated amino acid residue to a peptide. It was noted that, in this case, a free amino group in the analyte (i.e. a site of protonation) appeared essential for generating a MALDI response. A remarkable feature of this experiment is that it required photolysis in the solid-phase followed by sample diffusion into the matrix, protonation and transfer to the gas phase. In our hands, a similar experiment required the additional expediency of adding solvent to the photocleaved bead in order to
promote diffusion and subsequent cocrystallization of the analyte and matrix. Laser irradiation of the bead then afforded an excellent M A L D I / T O F signal. Library strategies that use high speed parallel compound synthesis to generate arrays of discrete compounds (as opposed to mixtures generated via the split resin approach) have been assuming greater significance in the drug discovery communit'y: Obtaining detailed analytical data on such product arrays is generally accepted as an important component of library quality control, and has placed increased demands on analysis throughput and data management. Several groups engaged in large scale parallel synthesis programs have described the integration of robotic synthesis systems with MS or LC-MS autosampling interfaces that permit MS data to be acquired on some or all of the compounds prepared in a synthesis run [29°,30,31]. Samples for which unsatisfactory data are obtained (e.g. estimated product yield below some threshold level) can be flagged for further attention. One analytical technique that has recently emerged as a potentially useful method for estimating library synthesis yields is H P L C with evaporative light-scattering detection (ELSD) [32]. In ELSD, the H P L C column effluent is mixed with a high-velocity stream of nitrogen gas and nebulized. T h e mobile phase is rapidly evaporated in a heated tube and the non-volatile solute is swept through a laser beam causing light scattering, which is detected by a photodiode. ELSD is more universal than UV detection; in general it detects any analyte above a molecular weight of 200 atomic cnass units. Another new development is the chemiluminescent nitrogen detector (CLND) for H P L C [33]. This destructive detector (which oxidizes nitrogen compounds to NO, then converts the N O to excited NO 2 by reaction with ozone and detects the emitted light in a photomultiplier) has been shown to give equivalent response per mole of N for all nitrogen compounds tested to date. It remains to be seen how robust this detector is in regular practice, but it offers the tantalizing promise of quantitative organic analysis of nitrogen containing compounds without purified reference samples for each analyte.
N M R spectroscopy N M R is the most powerful tool routinely used in solutionphase organic synthesis for structural characterization of small organic molecules. There has consequently been significant interest in the application of NMR spectroscopic methods for characterizing products of solid-phase synthesis and for monitoring the progress of chemical transformations on resins. Because the line widths in NMR spectra are greatly affected by both the mobility of atoms in a sample as well as tile homogeneity of the sample matrix, compound immobilization on solid
New
methods
for analyzing
supports dramatically influences the type and quality of spectral information that can be obtained by NMR.
on
polymeric
supports
Gallop
and
Fitch
97
phase synthesis of building blocks selectively enriched with I.~C enables the gel-phase method to be employed for convenient rapid reaction monitoring. Spectra with excellent signal-to-noise ratios can be obtained from -20 mg of resin in a 5 mm N M R tube in just a few minutes. Although the commercial availability of labeled starting materials is somewhat limited, we are frequently able to use this fast 13C N M R method for optimizing solid-phase synthetic protocols.
Early workers studying solid-phase peptide and oligonucleotide chemistry described the application of gel-phase muhinuclear N M R spectroscopy to the characterization of polymer-supported products [34]. In gel-phase NMR, a spectrum is acquired using a conventional liquids probe from a polymer sample that has been swollen in a suitable solvent. In comparison with solution-phase spectra, the signals obtained by the gel-phase method are relatively broad, and useful information is only obtained for nuclei such as ]3C, 31p and 19F which have wide spectral dispersion (for recent examples of applications of gel-phase 31p and 19F NMR, see [35-37]). Moreover, with gel-phase carbon NMR the low abundance of the 13C nucleus demands prolonged spectral acquisition times and the sample's spectrum may frequently be confounded by signals resulting from the polymer matrix. Gallop and co-workers [38,39] have described how the use in solidFigure
compounds
While 1H N M R is sufficiently sensitive to permit rapid analysis of solid-phase reactions, the signals obtained from resin samples by the gel-phase approach are typically too broad (>>25 Hz) to be useful in structure determinations. This line broadening can result from the restricted molecular mobility of the tethered compound, as welt as from magnetic field inhomogeneity surrounding the sample (susceptibility mismatches at the resin/solvent interface) and homonuclear dipolar interactions. These latter effects can be minimized by rapidly spinning the
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The 300 MHz MAS 1H NMR spectrum of TentaGel ~ resin-bound I}-Iactams. Note that the product is a 3:1 mixture of racemic cis lactams, with only the ~-configuration shown for convenience. Complete assignment of the lactam resonances has been made through a combination of COSY and decoupling studies. Several groups using MAS have successfully applied conventional two-dimensional NMR experiments (e.g. TOCSY, NOESY, 13C-1H HMQC) to resin samples using MAS [41,43,47,51].
98
Combinatorial chemistry
sample at the 'magic angle', and a number of recent reports have described how high-resolution 1H NMR spectra can bc obtained from resin-bound compounds using the magic angle sample spinning (MAS) technique [40-13]. Optimal spectra have been obtained from samples spun at - 2 k H z using very low volume (<40 p.L) NlklR probes (the Varian Nano-NMR probe) having careful susceptibility matching, although conventional solid state CP/MAS probes have been found to give good quality 1H NMR spectra also [44"]. Keifer [45"1 has published a detailed comparison of IN MAS spectra for the same organic sample attached to a range of synthesis resins using a variety of different solvents. These studies indicate that the resin structure is the dominant factor influencing line widths, with the solvent playing a secondary role. TentaGel ® resins, which have long, mobile polyethylene glycol tethers, generally provide the best line shapes, with line widths o f - 4 Hz being observed in CDzCIz. Each different type of resin (e.g. Wang, Merrifield, TentaGel @, etc.) gives a characteristic set of background signals, including broad aromatic resonances arising from rotationallv restricted nuclei in the polystyrene core. Presaturation or the application of spin-echo pulse sequences [46] are effective means of reducing these unwanted interferences, and can provide ill NMR spectra that are virtually equivalent in quality to those generated from soluble samples. An example of this type of high-resolution 1H MAS NMR spectrt, m, obtained from an isomeric mixture of monocyclic ]3-1actams produced via ketene cycloaddition to a resin bound imine, is shown in Figure 1 [47]. While high-resolution lit MAS spectra are routinely acquired in minutes from just 5 mg of resin, the sensitivity of the Nano-NMR probe is such that interpretable information can even bc obtained from a single resin bead. A 1-~C-filtered IH spectrum of (3,5-dimethoxy-l-~C) benzoic acid attached to a single bead of Wang resin (-800 pmol sample) clearly shows the expected doublet signal for the methoxy protons around 3.Tppm, though isotope editing was required to eliminate interferences from contaminants and solvent [48]. However, the limited amount of information that can be obtained from this type of single bead NMR analysis effectively makes the experiment a novelty More useful data have recently been obtained from single beads of a macro TentaGel~-type resin (400-750~m diameter. 10-65nmol sample) where direct 114 observation in a CP/MAS probe gave clear assignable resonances, and enabled intermediates in the synthesis of a hydantoin moiety to be readily characterized [49"]. Lin and Shapiro [50] have explored the use of pt, lsed-tield gradient technology with the TOCSY experiment as a means of obtaining useful NMR from mixtures of several compounds (e.g. a small combinatorial compound pool). This method assigns resonances on the basis of the diffusion coefficient for each proton in a moleculc, and
thus can distinguish resonances arising from different compounds. It is unclear how complex such mixtures can become before this techniqne is impractical. Conclusions
The mainstream analytical methods for organic structure elucidation, familiar from solution-phase synthesis, are rapidly being co-opted by solid-phase chemists. As the use of combinatorial techniques expands in areas as diverse as pharmaceutical research and new materials discover~, we can anticipate a dramatic increase in the number of reports of new solid-phase synthetic methods appearing in the scientific literature. The arbiters of quality control in organic chemistry determine which analytical methods will be used for proving the structure and purity of new organic compounds, and these standards are upheld through the editorial policies of chemical journals. While there are, as yet, no generally accepted standards for characterization of polymer-supported products, it would seem that appropriate analytical tools are becoming sufficiently widespread in the research community that formal analytical criteria could soon be established.
References
and
recommended
reading
Papers of particular interest, published within the annual period of review, have been highlighted as: * .-
of special interest of outstanding interest
1.
Hermkens PHH, Ottenheijm HCJ, Rees D: Solid phase organic reactions: a review of the literature. Tetrahedron 1996, 52:4527-4554.
2.
Fruchtel JS, Jung G: Organic chemistry on solid supports. Angew Chem /nt Ed 1996, 35:17-42.
3. Thompson LA, EIIman JA: Synthesis and applications of small • molecule libraries. Chem Rev 1996, 96:555-600. A review of small molecule library synthesis. Includes a useful compendium of analytical methods for solid-phase chemistry. 4.
Fitch WL (Ed): Analytical Methods for Quafity Control of Combinatorial Libraries, vol 1. Leiden: ESCOM Science Publishers; 1997, in press.
5.
Fitch WL, Detre G, Look GC: Combinatorial Chemistry and Molecular Diversity in Drug Discovery. Edited by Kerwin JF, Gordon EM. New York: John Wiley & Sons; 1997, in press.
6.
Schlick S, Bortel E, Dyrek K: Catalysis on polymer supports. Acta Po/ym 1996, 47:1-15.
7.
Crowley JI, Rapoport H: Solid-phase synthesis: novelty or fundamental concept? Accounts Chem Res 1976, 9:135-144.
8.
Yah B, Kumaravel G, Anjara H, Wu A, Petter RC, Jewell CF Jr, Wareing JR: Infrared spectrum of a single resin bead for realtime monitoring of solid-phase reactions. J Org Chem 1995, 60:5736-5738.
9. •,
Yan B, Fell JB, Kumaravel G: Progression of organic reactions on resin supports monitored by single bead FTIR microspectroscopy. J Org Chem 1996, 61:7467-7472. Very nice full paper, describing the use of single bead FTIR in a microscope instrument. Individual beads can be imaged under a microscope objective to provide spectra that approach the quality of those recorded on bulk resin samples. FTIR microscopy has excellent sensitivity and can detect strong IR chromophores (e.g. ester carbonyls) at the sub-picomole level. This method can also be useful for following the kinetics of polymer-supported reactions. 10.
Yan B, Gstach H: An indazole synthesis on solid support monitored by single bead FTIR microspectrocopy. Tetrahedron Lett 1996, 37:8325-8328.
New methods for analyzing compounds on polymeric supports Gallop and Fitch
11.
Russell K, Cole DC, McLaren FM, Pivonka DE: Analytical techniques for combinatorial chemistry: quantitative infrared spectroscopic measurements of deuterium-labeled protecting groups. J Am Chem Soc 1996. 118:7941-7945.
12.
Yan B, Kumaravel G: Probing solid-phase reactions by monitoring IR bands of compounds on a single 'flattened' resin bead. Tetrahedron 1996, 52:843-848.
13.
Gosselin F, Di Renzo M, Ellis TH, Lubell WD: Photoacoustic FTIR spectroscopy, a nondestructive method for sensitive analysis of solid-phase organic chemistry. J Org Chem 1996, 61:7980-7981.
14, •
Pivonski DE, Russell K, Gero T: Tools for combinatorial chemistry: in situ infrared analysis of solid-phase organic reactions. Appl Spectrosc 1996, 50:1471-1478. An impressive study describing a device for real time monitoring o1 solid phase reactions. Beads are trapped in an IR cell set up to allow reagents to flow through. FTIR spectra are then recorded and analyzed. Functional group changes can be monitored and optimized. 15.
Jung G, Beck-Sickinger AG: Multiple peptide synthesis methods and their applications, Angew Chem Int Ed 1992, 31:367-383.
16.
Carell T, Wintner EA, Sutherland AJ, Rebek J Jr, Dunayevskiy YM, Vouros P: New promise in combinatorial chemistry: synthesis, characterization, and screening of small-molecule libraries in solution. Chem Biol 1995, 2:1 ? 1-183.
17. •
Dunayevskiy YM, Vouros P, Carell T, Wintner EA, Rebek J Jr: Characterization of the complexity of small-molecule libraries by electrospray ionization mass spectrometry. Anal Chem 1995, 67:2906-2915. Study demonstrates utility of ESI-MS for analysis of soluble compound pools of moderate complexity.
99
Discusses practical considerations in establishing an automated high throughput system for mass spectrometric analysis of thousands of combinatorial samples per month in a pharmaceutical company environment. 30.
Garr CD, Peterson JR, Schultz L Oliver AR, Underiner TL, Cramer RD, Ferguson AM, Lawless MS, Patterson DE: Solutionphase synthesis of chemical libraries for lead discovery. J Biomol Screening 1996, 1:179-186.
31.
Cargill JF, Maiefski RR: Automated combinatorial chemistry on solid phase. Lab Robotics Automat 1996, 8:139-148.
32.
Kibbey CE: Quantitation of combinatorial libraries of small organic molecules by normal-phase HPLC with evaporative light-scattering detection, Mol Divers 1995, 1:24?-258.
33.
Fitch WL, Szardenings AK, Fujinari EM: Chemiluminescent nitrogen detection for HPLC: an important new tool in organic analytical chemistry. Tetrahedron Lett 1997, 38:1689-1692.
34.
Giralt E, Albericio F, Bardella F, Eritja R, Feliz M, Pedroso E, Pens M, Rizo J: Gel-phase NMR spectroscopy as a useful tool in solid-phase synthesis. In Innovation and Perspectives in SolidPhase Synthesis. Edited by Epton R. Birmingham: SPCC (UK) Ltd; 1990:111-120.
35.
Johnson CR, Zhang B: Solid-phase synthesis of alkene using the Horner-Wadsworth-Emmons reaction and monitoring by gel-phase 31p NMR. Tetrahedron Lett 1995, 36:9253-9256.
36.
Shapiro M J, Kumaravel G, Petter RG, Beveridge R: lSF Monitoring of a SNAr reaction on solid support. Tetrahedron Lett 1996, 37:4671-46?4.
3?.
Svensson A, Fex T, Kihlberg J: Use of 19F NMR spectroscopy to evaluate reactions in solid-phase organic synthesis. Tetrahedron Lett 1 996, 37:7649-7652.
38.
Look GC, Holmes CP, Chinn JP, Gallop MA: Methods for combinatorial organic synthesis: the use of fast 13C NMR analysis for gel-phase reaction monitoring. J Org Chem 1994, 59:7588-7590.
18.
Meutermans WDF, Alewood PF: The solid-phase synthesis of dihydro- and tetrahydroisoquinolines. Tetrahedron Lett 1995, 36:7709-7712.
19.
Gordon DW, Steele J: Reductive alkylation on a solid phase: synthesis of a piperazinedione combinatorial library. Bioorg Med Chem Lett 1995, 5:47-50.
39.
Hamper BC, Dukesherer DR, South MS: Solid-phase synthesis of proline analogs via a three component 1,3-dipolar cyeloaddition. Tetrahedron Lett 1996, 37:3671-3674.
Gordon EM, Gallop MA, Patel DV: Strategy and tactics in combinatorial organic synthesis. Applications to drug discovery. Accounts Chem Res 1996, 29:144-154.
40.
Parlow JJ, Normansell JE: Discovery of a herbicidal lead using polymer-bound activated esters in generating a combinatorial library of amides and esters. Mo/Divers 1995, 1:266-269.
Fitch WL, Detre G, Holmes CP, Shoolery JN, Keifer PA: High resolution 1H NMR in solid-phase organic synthesis. J Org Chem 1994, 59:7955-7956.
41.
Anderson RC, Jarema MA, Shapiro MJ, Stokes JP, Ziliox M: Analytical techniques in combinatorial chemistry: MAS CH correlation in solvent-swollen resin. J Org Chem 1995, 60:2650-2651.
42.
Wehler T, Westman J: Magic angle spinning NMR: a versatile tool for monitoring the progress of solid phase synthesis. Tetrahedron Lett 1996, 37:4771-4774.
43.
Pop IE, Dhalluin CE Deprez BP, Melnyk PC, Lippens GM, Tartar AL: Monitoring of a three step solid phase synthesis reaction involving a Heck reaction using magic angle spinning NMR spectroscopy. Tetrahedron 1996, 52:12209-12222.
20.
21.
22.
Parlow JJ: The use of anionic exchange resins for the synthesis of combinatorial libraries containing aryl and heteroaryl ethers. Tetrahedron Lett 1996, 37:5257-5260.
23.
Zambias RA, Boulton DA, Griffin PR: Microchemical structural determination of a peptoid covalently bound to a polymeric bead by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, Tetrahedron Lett 1994, 35:4283-4286.
24.
Brummel CL, Lee INW, Zhou Y, Benkovic SJ, Winograd N: A mass spectrometric solution to the address problem of combinatorial libraries, Science 1994, 264:399-402.
25.
Brown BB, Wagner DS, Geysen HM: A single bead decode strategy using electrospray ionization mass spectrometry and a new photolabile linker. Mo/Divers 1995, 1:4-12.
26.
Egner BJ, Langley G J, Bradley M: Solid-phase chemistry: direct monitoring by matrix-assisted laser desorption/ionization timeof-flight mass spectrometry. A tool for combinatorial chemistry. J Org Chem 1995, 60:2652-2653.
27. •
Brummel CL, Vickerman JC, Carr SA, Hemling ME, Roberts GD, Johnson W, Weinstock J, Gaitanopoulos D, Benkovic SJ, Winograd N: Evaluation of mass spectrometric methods applicable to the direct analysis of non-peptide bead-bound combinatorial libraries. Anal Chem 1996, 68:237-242. A comparison of ESI-MS, MALDI/TOF-MS and TOF/SIMS for characterization of the material from a single bead of a combinatorial library. Fitzgerald MC, Harris K, Shevlin CG, Siuzdak G: Direct characterization of solid phase resin-bound molecules by mass spectrometry. Bioorg Med Chem Lett 1996, 6:979-982. Direct MALDI of a peptide ligand on Wang resin with a photolinker. MALDI provides both photoc}eavage and ionization.
44. •
Keifer PA, Baltusis L, Rice DM, Tymiak AA, Shoolery JN: A comparison of NMR spectra obtained for solid-phasesynthesis resins using conventional high-resolution, magicangle-spinning and high-resolution magic-angle-spinning probes. J Magn Res 1996, 119:65-75. A comparison of the Nano-NMR probe with CP/MAS and conventional probes for 1H and 130 spectra of solid-phase resins and tethered molecules. 45. ••
Keifer PA: Influence of resin structure, tether length, and solvent upon the high-resolution 1H NMR spectra of solid phase synthesis resins. J Org Chem 1996, 61:1558-1559. An interesting comparison of 1H MAS NMR data obtained for several protected aspartic acid residues attached to a variety of commercially available resins. The most important factors influencing proton line-widths are resin structure and the NMR solvent, with polyethyleneglycol-grafted resins like TentaGel ~';swollen in CD2CI 2 affording the best resolution. 46.
28. •
29. •
Hegy G, Gorlach E, Richmond R, Bitsch F: High throughput electrospray mass spectrometry of combinatorial chemistry racks with automated contamination surveillance and results reporting. Rapid Commun Mass Spectrom 1996, 10:1894-1900.
Garigipati RS, Adams B, Adams JL, Sarkar SK: Use of spin echo magic angle spinning 1H NMR in reaction monitoring in combinatorial organic synthesis. J Org Chem 1996, 61:2911-2914.
47
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