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Figure 13 MassLynx (Micromass UK Limited, Manchester, UK) OpenLynx open access browser data report. Plate configuration is shown in upper left. MS and UV data is displayed for the selected well position. If the requested mass is found, the well position is highlighted in green. If it is not, it is highlighted in red. Browser’s of this type are also used to track fractions in mass directed auto-purification systems.

are also used in this configuration. The upstream splitter divides the preparative flow and is an integral part of the system. The second splitter splits the analytical flow for parallel MS and PDA detection.

The use of an upstream splitter as outlined in Fig. 14 has several distinct advantages. Besides being easy to use and reproducible, this type of splitter provides constant delay times across a wide flow range, without the need for plumbing different tubing lengths or diameters. The use of a makeup flow allows high column loading without saturating the detector (by dilution) and allows flow to be split to multiple detectors without back streaming (explained below). The splitter essentially has two sides: a high-pressure side (column to collector) and a low-pressure side (makeup to detectors). As long as this balance is maintained the system works correctly. In operation, however, as the gradient composition changes from aqueous to organic, the back pressure

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Figure 14 High-throughput mass-directed auto purification system schematic. System as shown is configured for preparative two-column regeneration and an analytical column for method development or to reanalyze fractions.

in the system decreases. Eventually the gradient back pressure can fall below the constant pressure of the makeup pump, resulting in splitter back streaming which prevents sample from getting to the MS probe and prevents late eluting peaks from being seen or collected. By using a lower viscosity organic solvent (e.g., 100% MeOH) as the makeup solvent, the back pressure is reduced to below that of the lowest point in the gradient, and back streaming is prevented. Subtle variations in split ratio delay times and band broadening are nominal across all gradient compositions.

Mass-directed autopurification systems are designed so that a peak is detected at the MS prior to reaching the fraction collector. Different flow rate– dependent delay times must be taken into account, and the software controls the synchronization between detection and collector trigger times. Figures 15 and 16 highlight the results of a mass-directed autopurification experiment (22). In this experiment, a three-component synthetic mixture was fractionated on the preparative scale and the fractions reanalyzed on the same system. Figure 15 shows the total ion chromatogram, UV signal, and fraction collection signal for the 20 mg/mL preparative run. As can be seen from the fraction collection signal, as the chromatography changes, the intelligent fraction collection compensates. The MS response directed the fraction collection based on the molecular weight of the three components in each instance. Figure 16 depicts the reanalysis of the second fraction on the analytical scale, on the same system. In spite of the two closely eluting peaks before and after, the data show a clean spectrum free of contamination.

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Figure 15 Mass directed fraction collection. Separation was performed on Waters Auto-purification System with FractionLynx software (Waters Corporation, Milford, MA) on a 19 by 50mm 7 micron particle size Symmetry C18 column at ambient

temperature. The mobile phase consisted of 0.1% phosphoric acid as the A solvent, and acetonitrile as the B solvent, run as a linear gradient from 0–100% B over 8 minutes at 20.0 mL/min. UV detection at 254nm, and a 10 mL injection was used. Peaks (20 mg/mL each in 50/50 methanol/water) in order of elution are terfenadine, diphenhydramine, and oxybutynin chloride. The two lower traces show the MS total ion chromatogram (TIC-lower left in figure-generated with an electrospray interface), and the UV trace (lower right, an extracted PDA channel) for five separate overlaid runs. A separate mass directed fraction collection signal for each of the five runs is shown in the upper right. Note the timing differences—as the preparative chromatography changes, the intelligent fraction collection compensates.

V.THE FUTURE OF LIQUID CHROMATOGRAPHY IN COMBINATORIAL CHEMISTRY

As stated earlier, LC has emerged as the technique of choice for assessing the quality of combinatorial libraries. While this trend will certainly continue, LC, either alone, or in combination with other techniques, will continue to evolve. The trend towards shorter, smaller internal diameter columns will continue, however hardware limitations such as pressure limitations, system volume, injection overhead times and data storage must be overcome. A balance be-

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Figure 16 Analytical scale reanalysis of fraction two from Figure 14. The presence of a single compound (oxybutynin, m/z 357.3) on the TIC (top) is confirmed by the UV data (bottom), and the MS on the right lacks evidence of contamination from peaks one and three in the preparative run. Separation conditions that closely match those reported in Figure 2B were used.

tween cycle to cycle analysis times and peak capacity, whether obtained by an actual separation or by instrument specificity or deconvolution must be maintained. Recent reports of the use of capillary LC may also play a significant role in new developments in the field of combinatorial chemistry (23). Capillary LC is currently used mainly for sensitivity enhancements. Chromatographic theory predicts that reducing column diameter results in a mass sensitivity increase that is inversely proportional to the square of the column diameter ratios. However, as mentioned previously, but especially on the capillary scale, extra care should be taken to minimize extra column band broadening by using reduced diameter tubing, smaller volume detector cells, and proper fitting connections (9). In addition, capillary LC has a distinct advantage over analytical scale LC when interfaced to MS. Using analytical systems, sensitivity is sacrificed either by introducing the entire sample into the MS interface and losing much of the sample by high capacity pumping or by flow splitting just before the MS inlet. Low microliter per minute capillary flows are appropriate for direct interfacing, so virtually the entire sample is introduced into the MS ion source. Additional high throughput capillary LC techniques and methods currently in development may eventually extend the use

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of this technique to combinatorial applications (24). Miniaturization of different sorts is also under way that may eventually find LC applications in combinatorial chemistry. Microelectromechanical systems, or MEMS, or other ‘‘chip-based’’ technology may eventually be optimized for use in screening applications in the drug discovery process.

Detection schemes will also continue to evolve. While NMR is covered in another chapter in this volume, advances in NMR used as an LC detector have also been reported (25–28) that may eventually increase the utility of this tool for combinatorial chemistry. Used in both stopped flow and on line modes, LC/NMR can be extremely useful for structure elucidation provided the proper mobile phases or solvent suppression techniques are used (27).

Affinity chromatographic techniques are also finding applications. For example, peptide ligands can be immobilized on chromatographic media that will selectively bind and release target molecules under optimized conditions (29, 30). Stable, selective, high affinity ligands used in this manner can purify a target molecule from a complex mixture, even in the presence of closely related impurities. It is possible to increase or modulate the strength of binding and incorporate chromatographic selectivity against specific contaminants, which can accelerate process development and increase product recovery in drug discovery.

VI. CONCLUSION

LC characterization of combinatorial libraries is a vital tool in drug discovery, and, in one format or another, will remain so for a long time. High throughput methods, open access environments, mass directed auto-purification, and total system integration have lead to increased utilization of LC in ways that a few short years ago were unimaginable. This trend can only continue, as researchers strive for higher throughput, and wider applicability in solving challenges in drug discovery.

ACKNOWLEDGMENTS

The author would like to acknowledge the contributions of Chris Chumsae, Andrew Brailsford, Jeffrey Holyoke, and Beverly Kenney of Waters Corporation who collaborated in various ways in the data generated for use in this chapter.

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REFERENCES

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2.JN Kyranos and JC Hogan, Jr. Anal Chem 70(11):389A–395A, 1998.

3.WK Goetzinger and JN Kyranos. Am Lab April:27–37, 1998.

4.R Cole, KA Laws, DL Hiller, JP Kiplinger, and RS Ware. Am Lab July:15–20, 1998.

5.ME Swartz, M Balogh, and B Kenney. Poster presented at the Eastern Analytical Symposium, Somerset NJ, November 1998, and Waters Corporation (Milford, MA) Literature Code M29.

6.JP Kiplinger, RO Cole, S Robinson, EJ Roskamp, RS Ware, HJ O’Connell, A Brailsford, and J Batt. Structure controlled automated purification of parallel synthesis products in drug discovery. Rapid Commun Mass Spectrom 12:658–664, 1998.

7.L Zeng, L Burton, K Yung, B Shushan, and DB Kassel. Automated analytical/ preparative HPLC-MS system for the rapid characterization and purification of compound libraries. J Chromatogr A 794:3–13, 1998.

8.HN Weller, MG Young, SJ Michalczyk, GH Reitnauer, RS Cooley, PC Rahn, DJ Loyd, D Fiore, and SJ Fischman. Mol Div 3:1–24, 1997.

9.LR Snyder, JJ Kirkland and JL Glajch. Practical HPLC Method Development. Second Edition. New York: John Wiley and Sons, Inc., 1997.

10.JL Li and J Morawski. LC/GC Magazine 16(5):468–476, 1998, and Waters Corporation (Milford, MA) Literature Code Number T158.

11.C Chumsae and A Brailsford. Poster presented at ASMS, Houston, TX. June 1999.

12.Brochure No. BR25/DAM, Micromass Corporation, Manchester UK, April 1999.

13.A Stolyhwho, H Colin, and G Guichon. J Chromatogr 265:1–18, 1983.

14.A Stolyhwho, H Colin, M Martin, and G Guichon. J Chromatogr 288:253–275, 1984.

15.TH Mourey and LE Oppenheimer. Anal Chem 56:2427–2434, 1984.

16.M Lafosse, C Elfakar, L Morin-Allory, and M Dreux. J High Res Chromatogr 15:312–318, 1992.

17.CE Kibbey. Mol Div 1:247–258, 1995.

18.A Brailsford and C Chumsae. Poster presented at ASMS, Houston TX. June 1999.

19.EW Taylor, MG Qian, and GD Dollinger. Anal Chem 70:3339–3347, 1998.

20.R Bizanek, JD Manes, and EM Fujinari. Peptide Res 8:40–44, 1996.

21.WL Fitch, AK Szardenings, and EM Fujinari. Terahedron Lett 38:1689–1692, 1997.

22.ME Swartz and BF Kenney. Poster presented at the Eastern Analytical Symposium, Somerset, NJ, November 1998.

23.SA Cohen, BF Kenney, J Holyoke, TA Dourdeville, and D Della Rovere. LC/ GC, 17(4S):S9–S16, 1999.

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24.ME Swartz. Unpublished results, 1999.

25.PA Keifer. DDT, 2(11):468–478, 1997.

26.FS Pullen, AG Swanson, MJ Newman, and DS Richards. Rapid Comm Mass Spectr 9:1003–1006, 1995.

27.SH Smallcombe, SL Patt, and PA Keeifer. J Mag Res, Series A 117:295–303, 1995.

28.H Barjat, GA Morris, MJ Newman, and AG Swanson. J Mag Res, Series A 119: 115–119, 1996.

6

Capillary Electrophoresis in Combinatorial Library Analysis

Ira S. Krull

Northeastern University

Boston, Massachusetts

Christina A. Gendreau

Waters Corporation

Milford, Massachusetts

Hong Jian Dai

Shuster Laboratories

Quincy, Massachusetts

I.INTRODUCTION AND OVERVIEW: WHY HPCE FOR COMBINATORIAL MAPPING ANALYSIS?

Although capillary electrophoresis (CE),1 also known as high-performance CE or HPCE, has been known and described in the literature for almost two decades, its use for combinatorial mapping is much more recent (1–14). There does not appear to be a previous review, other than that in Analytical Chemistry (an American Chemical Society journal), that describes the general applicability and applications of CE for these purposes (15). There are relatively few actual publications in the refereed literature that have utilized various CE modes to perform analysis of combinatorial maps. At the same time, there are

1 All acronyms and abbreviations are defined in the glossary preceding the references.

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many more CE papers that have used affinity recognition to identify an active antigen or antibody or receptor molecule, but not as part of a larger library of similarly structured compounds. Most papers in the literature deal with the synthesis and characterization of libraries in terms of chemical structures, not biological activity (15–17). Because sections of this book are devoted to analytical approaches to screen compounds in combinatorial libraries for activity, it represents the first major effort to describe how analytical chemistry can be applied to identify individual, active members of a chemical library. In this chapter, the focus is on describing how CE approaches (methods, instrumentation, protocols, techniques) can be applied to a combinatorial library and isolate, as well as characterize, only those active (lead) compounds in a given library. This is quite different from characterizing all of the members of a given library in terms of their individual structures or saying that a given structure is present in a particular library.

In general, CE is perhaps ideally situated for combinatorial map searching, as well as for providing structural information about lead/target compounds in that library. Any useful analytical approach, be it mass spectrometry (MS), nuclear magnetic resonance (NMR), Fourier transform infrared (IR), high-performance liquid chromatography (HPLC), or HPCE, must accomplish several ideal goals: (a) recognize those compounds that shall prove of biological interest and opportunity, using some type of molecular/biological recognition (antibody–antigen, receptor binding protein–antigen, drug–protein, and so forth); (b) separate the recognized members of the library from those showing little or no biological interactions with biopolymers or target drugs; (c) separate active, lead compounds from one another on some reasonable time scale; (d) indicate general level of molecular recognition (high vs. low); and

(e) provide some type of structural information about the active compounds, using techniques such as NMR, MS, and FTIR. Quantitation of lead or active compounds is not really necessary for any library, since presumably one can make more of those interesting compounds at a later date, once they are shown to be recognizable and structurally determined. The successful analytical technique is more qualitative than quantitative, but ideally it will provide an indication of biological activity and structural information. One without the other is less than ideal for a useful, practical, and valuable analytical method of mapping a combinatorial library.

We use the word mapping here not so much to identity all of the structures present but rather to identity the structures of just those active (target, potential leads) compounds in that library. It has become difficult to determine the structures of individual members of a large, complex library; it is much easier to pinpoint which members of that library are indeed biologically active

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and then determine their individual structures. Although analytical techniques have been used mostly to identify potential pharmaceutical agents from a combinatorial library, it should be clear that these methods could be applied for virtually any purpose, so long as one has a recognition element or compound available. That is, libraries can be applied for the identification of agricultural chemicals, flavors, perfumes, fragrances, insecticides, insect pheromones, and so forth. What is crucial for the success of these methods really depends on the recognition element, and the process by which a compound or compounds will indicate biological or chemical activity for the stated purposes.

CE is potentially an ideal approach for mapping of combinatorial libraries because it is a purely liquid phase technique, as opposed to HPLC, which requires some sort of solid–liquid interaction, partitioning, diffusion, etc. There are arguments against using solid phase techniques, such as immunoaffinity chromatography (ICA) or high-performance affinity chromatography (HPAC) to isolate active, lead compounds, rather than a purely liquid solution approach. As Dunayevskiy has aptly described and discussed, solid phase binding assays, such as using immobilized antibodies to identify active antigens in HPAC or immunodetection (ID), can be influenced by the site or nature of the attachment of the ligand to the solid support (18).

This is a classic problem in all immunorecognition or immunodetection or affinity isolation techniques in HPLC or, in the future, in capillary electrochromatography (CEC). The support (bead) used to hold the ligand or receptor molecules in any affinity recognition framework can present conformational limitations that can at times prevent any or all binding to the ligand or receptor. Unfortunately, there can be no firm rule for the optimal offering of a compound to a receptor or vice versa. There are a large number of ways to immobilize a receptor protein or its ligand, but none of these will guarantee that the bound species can now be adequately recognized and captured by its partner (ligand receptor) (19–35). The area of immobilization of compounds to a solid support is immense, with a large number of linkage agents commercially available. It is never fully clear which of these will provide a bound species offering itself in the optimal way to its partner, now in a solution passing through that bed. It is always possible to use more than one method of immobilization of the same receptor protein in the hope that this will improve the chances of capturing any and all possible partners/ligands (19,21). However, these methods only approximate complete freedom from steric effects, and one is left with limits on 100%, true recognition interactions that occur in a purely solution phase recognition approach. For these very reasons, more and more applications of combinatorial library searching are being described using purely solution phase approaches.