Single Bead Characterization Using Analytical Constructs: Application

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Single Bead Characterization Using Analytical Constructs: Application to Quality Control of Libraries Olivier Lorthioir,*,† Robin A. E. Carr,† Miles S. Congreve,‡ Mario H. Geysen,§ Corinne Kay,‡ Peter Marshall,† Stephen C. McKeown,*,† Nigel J. Parr,† Jan J. Scicinski,‡ and Stephen P. Watson†

Glaxo SmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, U.K., Glaxo SmithKline-Cambridge Chemistry Laboratory, University Chemical Laboratories, Lensfield Road, Cambridge, CB2 1EW, U.K., Glaxo SmithKline, Research Triangle Park, North Carolina 27709

Analytical construct technology has been successfully applied to the single-bead analysis of a split-mix combinatorial library. Substrates can be released from the resin by conventional cleavage for biological screening. Alternatively, for the purpose of analysis and quality control, cleavage at an orthogonal construct linker produces an analytical fragment incorporating the substrate. This analytical fragment is highly sensitized to electrospray mass spectrometry (ESI-MS) and is easily identified by isotope labeling. The construct cleavage rendered readily visible even those compounds that clearly could not be seen by conventional cleavage and mass spectrometry analysis. A 1H NMR control experiment proved that the compounds cleaved conventionally were, however, present in the sample in good yield and purity. In view of the data obtained, we think that this is a significant and important step toward solving our current quality control problems. The increasing demand for new therapeutic agents has been the driving force for the recent acceptance and integration of combinatorial techniques in drug discovery companies. Combinatorial methods aim at producing large collections of well-defined compounds (so-called libraries) in a relatively short time. Most combinatorial libraries are synthesized by means of the solid-phase chemistry, which has many advantages when compared to traditional solution-based approaches.1-4 Mainly, reactions can be driven to completion by use of excess reagents, the latter, and byproducts not bound to the solid support being removed simply by washing. The solid support usually takes the form of small polymeric beads, and one widely adopted strategy to produce libraries is the “split and mix” approach, in which each individual resin bead contains a single library compound at the end of the multiple step synthesis.5-7 * Corresponding authors: Fax: +44 (0) 1438 76 3616. E-mail: ol65917@ gsk.com; [email protected]. † Medicines Research Centre. ‡ Cambridge Chemistry Laboratory. § Research Triangle Park. (1) Thompson, L. A.; Ellman, J. A. Chem. Rev. 1996, 96, 555-600. (2) Balkenhohl, F.; von dem Busche-Hunnefeld, C.; Lansky, A.; Zechel, C. Angew. Chem., Int. Ed. Engl. 1996, 35, 2288-2337. (3) Fruchtel, J. S.; Jung, G. Angew. Chem., Int. Ed. Engl. 1996, 35, 17-42. (4) Lam, K. S.; Lebl, M.; Krchnak, V. Chem. Rev. 1997, 97, 411-448. 10.1021/ac000814y CCC: $20.00 Published on Web 01/27/2001

© 2001 American Chemical Society

One can argue that characterization of a library in order to assay the success of the synthesis before its screening against the biological target is unnecessary and that only the active compound needs to be characterized. However, this philosophy can lead to overestimation of the diversity explored, false negatives (compound not present on the bead), and false positives (unexpected but irreproducible active side product). Quality control (QC) of libraries prior to screening is therefore essential to avoid these type of misleading results: such a library analysis, by verifying the composition and confirming the explored diversity, enables the reliable gathering of results in the following biological screening. However, quality control of one bead/one compound libraries remains a challenge for analytical chemists.8,9 First, the amount of compound available is limited. For instance, a 140-µm ArgoGel bead provides a theoretical loading of 700 pmol/bead. However, the actual loading is often less, due to variations in bead size, overall synthesis yield, and final cleavage efficiency. Second, although total characterization of a library is not realistic, a large number of beads must nevertheless be analyzed in order to provide meaningful data for library quality evaluation. As a consequence, the analytical technique that is used must be generic and amenable to automation for high throughput. High-throughput ESI-MS,10-13 by far the most commonly used of the analytical techniques available for the characterization of combinatorial libraries,14-17 is now well-implemented in pharma(5) Furka, A.; Sebestyen, F.; Asgedom, M.; Dibo, G. Int. J. Pept. Protein Res. 1991, 37, 487-493. (6) Houghten, R. A.; Pinilla, C.; Blondelle, S. E.; Appel, J. R.; Dooley, C. T.; Cuervo, J. H. Nature 1991, 354, 84-86. (7) Lam, K. S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J.; Kazmierski, W. M.; Knapp, R. J. Nature 1991, 354, 82-84. (8) Murray, R. W. Anal. Chem. 1997, 5755 A. (9) Czarnik, A. W. Anal. Chem. 1998, 378, 8A. (10) Sussmuth, R. D.; Jung, G. J. Chromatogr. B 1999, 725, 49-65. (11) Loo, J. A. Eur. Mass Spectrom. 1997, 3, 93-104. (12) Swali, V.; Langley, G. J.; Bradley, M. Curr. Opin. Chem. Biol. 1999, 3, 337341. (13) Brummel, C. L.; Vickerman, J. C.; Carr, S. A.; Hemling, M. E.; Roberts, G. D.; Johnson, W.; Weinstock, J.; Gaitanopoulos, D.; Benkovic, S. J.; Winograd, N. Anal. Chem. 1996, 68, 237-242. (14) Egner, B. J.; Bradley, M. Drug Discovery Today 1997, 2, 102-109. (15) Gallop, M. A.; Fitch, W. L. Curr. Opin. Chem. Biol. 1997, 1, 94-100. (16) Fitch, W. L. Mol. Diversity 1999, 4, 39-45.

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Figure 1. Alternative cleavages of an analytical construct resin.

ceutical industries.18,19 However, as a consequence of the inherent great structural diversity of small organic compound libraries, many samples do not have the appropriate ionization properties for mass spectrometry to be universally applicable. So, as powerful as ESI-MS proved to be, in our laboratory, we are still having problems with some drug-like molecules that exhibit no “MSfriendly” properties, especially for single-bead analysis. Techniques are available that potentially address some specific issues (e.g., use of negative scan mode to analyze acid-containing compounds). However, we required a generic method that would tackle even problematic neutral species containing neither basic nor acidic groups. Recently, analytical constructs have been introduced to address such problems of analysis by mass spectrometry.20,21 A typical analytical construct (Figure 1) contains a linker, allowing the release of substrates in the conventional manner, linker 2, but also an orthogonal linker, linker 1, cleavable by a specific reagent in an additional analytical mode. If the analytical mode is used, the released fragment contains an amine that enhances electrospray ionization properties and an isotope label that facilitates identification of relevant signals (appearing as characteristic doublets) in the mass spectrum. These dual-linker analytical constructs have been designed to address problems of monitoring and analysis of reactions conducted on solid support and have been reported to reliably render all compounds visible to mass spectrometry, even from single bead.20 In the present work, we report the application of the analytical construct methodology to the high-throughput analysis of samples derived from single beads. A proof of concept study using a 64member secondary amide library demonstrates how this generic approach could facilitate quality control of any bead-based library. The single-bead ESI-MS data that was collected allowed for a direct comparison between analysis conducted after conventional cleavage and analysis using analytical construct cleavage. The experiment clearly demonstrated the advantages of the latter methodology. EXPERIMENTAL SECTION Preparation of Construct Resin 1. The synthesis of the thiopyrimidine construct is described elsewhere.22 Resin 1 (Figure 3) was prepared as follows. The thiopyrimidine construct acid (3 (17) Fitch, W. L.; Look, G. C.; Detre, G. Comb. Chem. Mol. Diversity Drug Discovery 1998, 349. (18) Lewis, K. C.; Fitch, W. L.; Maclean, D. LC-GC 1998, 16, 644-649. (19) Hegy, G.; Gorlach, E.; Richmond, R.; Bitsch, F. Rapid Commun. Mass Spectrom. 1996, 10, 1894-1900. (20) Lorthioir, O.; McKeown, S. C.; Parr, N. J.; Washington, M.; Watson, S. P. Tetrahedron Lett 2000, 41, 8609-8613.

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equiv), HATU (3 equiv), and DIPEA (6 equiv) were dissolved in DMF (solution 0.15 M in construct) and left to stand for 10 min. This solution was then added to ArgoGel resin (0.4 mmol/g loading, 1 equiv) and shaken overnight. The resin was drained and washed with DMF, DCM, Et2O, DCM, and finally, Et2O, then dried in vacuo. A solution of 20% TFA in DCM was then added to the resin and shaken for 2 h. The resin was drained, washed as above, and then shaken with 10% DIPEA in DMF for 10 min. The resin was drained, washed as above, and dried in vacuo. A solution of the acid-labile linker that was used (monomethoxy aldehyde, 5 equiv), DIPEA (10 equiv), and PyBOP (5 equiv) in DMF (solution, 0.15 M in monomethoxy aldehyde linker) was added to the resin, and the resin was shaken overnight. The resin was drained and washed as above, then dried in vacuo to give resin 1 (Figure 3). Model Library Synthesis. Resin 1 was then distributed into 64 small polypropylene reactors (25 mg resin/reactor) (MicroKans, IRORI system).23,24 A radio frequency ID tag was then inserted into each MicroKan before sealing it. A computer database assigning each reactor to a member of the final library was created, thus encoding the library. At any step during the synthesis, each reactor was identified by reading its ID tag and put into the correct vessel for the subsequent reaction. Step 1: Resin Attachment of the First Monomer (Amine). To each of 4 vessels containing 16 IRORI MicroKans (1 equiv, 120 µmol) (each can containing 25 mg, 7.5 µmol of resin 1) in DCM (30 mL) was added a premixed solution of the appropriate amine (R1: A, B, C, or D; Table 1, Figure 4) (20 equiv) and acetic acid (20 equiv) in DCM (2 mL), and the mixture was shaken for 1h. To each vessel was then added a preformed mixture of tetrabutylammonium borohydride (20 equiv) and acetic acid (20 equiv) in DCM (5 mL), and the MicroKans were shaken overnight. The vessels were then drained and the MicroKans were washed with DCM, DMF, DCM, Et2O, DCM, and Et2O. Step 2: Coupling of the Second Monomer (Amino Acid). To each of 4 vessels containing 16 IRORI MicroKans in DMF (30 mL) (after MicroKan distribution using ID tags) was added a solution of PyBOP (10 equiv), DIPEA (20 equiv), and one of the Fmocprotected amino acids (R2: E, F, G, or H; Table 1, Figure 4) (10 equiv) in DMF (13 mL), and the MicroKans were shaken overnight. The vessels were then drained, and the MicroKans were washed as above. To a vessel containing all 64 MicroKans was then added a solution of piperidine (20%) in DMF, and the MicroKans were shaken for 3h. The vessel was then drained and the MicroKans were washed as above. Step 3: Coupling of the Third Monomer (Carboxylic Acid). To each of 4 vessels containing 16 IRORI MicroKans in DMF (30 mL) (after MicroKan distribution using ID tags) was added a solution of PyBOP (10 equiv), DIPEA (20 equiv), and one of the acids (R3: I, J, K, or L; Table 1, Figure 4) (10 equiv) in DMF (5 mL), and the MicroKans were shaken for 4 h. The vessels were then drained, and the MicroKans were washed as above. Single-Bead TFA Cleavage, Method 1. (For explanation of Methods 1 & 2, see Results and Discussion.) Single beads from each of the 64 MicroKan reactors were distributed into individual (21) Nicolaou, K. C.; Xiao, X.-Y.; Parandoosh, Z.; Senyei, A.; Nova, M. P. Angew. Chem., Int. Ed. Engl. 1995, 34, 2289. (22) Moran, E. J.; Sarshar, S.; Cargill, J. F.; Shahbaz, M. H.; Lio, A.; Mjalli, A. M. M.; Armstrong, R. W. J. Am. Chem. Soc. 1995, 117, 10787.

Figure 2. Analytical construct resin used in this study. Route a, analytical fragment release; route b, classical cleavage for biological screening.

Figure 3. Preparation of the construct resin 1.

wells of a 96-well bead cup plate (experiment performed in duplicate). Bead cup plates are modified microtiter plates that have been pierced to allow liquid to be spun through a hole and yet be small enough to retain the bead in the well.25 The beads can be washed with solvent that is removed by centrifugation and are then dried in a vacuum centrifuge. TFA cleavage of compounds from the resin can be carried out in the well. The hole is small enough that cleavage medium is retained in the well until the plates are placed in a centrifuge. After cleavage, the medium is spun through into a collection plate. 15 µL of TFA/H2O 9/1 were added to each well and allowed to stand for 4 h. After centrifugation and washing of the beads with 15 µL of TFA/H2O 9/1, the plate was placed in a vacuum (23) Cox, B.; Denyer, J. C.; Binnie, A.; Donnelly, M. C.; Evans, B.; Green, D. V. S.; Lewis, J. A.; Mander, T. H.; Merritt, A. T.; Valler, M. J.; Watson, S. P. Prog. Med. Chem. 2000, 37, 83.

centrifuge, and the samples were evaporated to dryness in 30 min. After being reconstituted with 15 µL of DMSO the samples were analyzed using ESI-MS. Single Bead TFA Cleavage, Method 2. Single beads from each of the 64 MicroKans reactors were distributed into individual wells of a 96-well microtiter plate (experiment performed in duplicate). 15 µL of TFA/H2O 9/1 were added to each well, and the plate was allowed to stand for 4 h. The plate was then directly placed in a vacuum centrifuge, and the samples were evaporated to dryness in 30 min. After being reconstituted with 15 µL of DMSO, the samples were analyzed by ESI-MS. Single Bead Analytical Construct Cleavage. Single beads from each of the 64 MicroKan reactors were distributed into individual wells of a 96-well bead cup plate (experiment performed in duplicate). 15 µL of OXONE (0.1 M in water) were added to each well and the plate was allowed to stand for 1 h. The plate Analytical Chemistry, Vol. 73, No. 5, March 1, 2001

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Figure 4. Scheme of the reactions used in library synthesis.

Table 1. Set of Monomers Used in Library Synthesis

scanning a m/z 100-850 (or 150-1500) window in 1 s. The total analysis time (time between injections) for each sample was 10 min. An Ultra plus pump (Microtech Scientific) was used to deliver a water-acetonitrile gradient at 25 µL/min. The mobile phase was composed of, A, water (0.1% formic acid) and, B, 95% acetonitrile (0.05% formic acid) and was delivered in the following gradient: 0 min, 5% B; 0.1min, 5% B; 2 min, 100% B; 7.5 min, 100 %; 7.6 min, 5% B. An Autosampler CTC Analytics was used to inject 10 µL of sample from the microtiter plate. A 5 cm × 0.32 mm Zorbax C18 SB media column (Microtech Scientific), with a particle size of 5 µm, was used. TFA Cleavage on a Bulk Quantity of Resin. After lid removal, each of the 64 MicroKans (containing about 22 mg of resin) was placed in a filter tube, 2 mL of a TFA/H2O mixture (9/1) were added, and the filter tube shaken for 4 h. The resin was then drained, washed with 1 mL TFA/H2O 9/1, and the pooled filtrates were evaporated to dryness. The residual solid or oil was then dissolved in 800 µL of DMSO-d6 to obtain the 1H NMR spectrum. The overall yield was calculated on the basis of an initial resin loading of 0.28 mmol/g, using integration relative to the DMSO-d5 peak.

(a) Monomers B and C as free side chains after TFA deprotection.

was then placed in a centrifuge to remove the OXONE solution. After washing the beads with 15 µL of water (2 times) and 15 µL of methanol, 15 µL of 1-methylpiperazine (10%) in DMSO was added to each well, the plate was covered with a lid, and it was allowed to stand overnight. After centrifugation, the collected samples were analyzed using ESI-MS. MS Analysis. The MS analysis was performed on a Finnigan LCQ ion trap mass spectrometer in positive electrospray mode, 966

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RESULTS AND DISCUSSION 1) Design and Synthesis of a Library Modeling Split Mix Strategy Quality Control Issues. (i) Experimental Design. The thiopyrimidine analytical construct (Figure 2) is one of the linkers that was dedicated to tackle analysis problems on solid support and has been described elsewhere.22 It was selected for the present study in view of its compatibility with the chemical transformations envisaged. Analytical cleavage is effected by nucleophilic displacement using 1-methylpiperazine after activation by OXONE oxidation of the alkylthio linkage, providing a safety-

Table 2. Summarized Data for All 64 Library Components

library memberb

single-bead conventional analysisa method 1c method 2c

AEI AEJ AEK AEL AFI AFJ AFK AFL AGI AGJ AGK AGL AHI AHJ AHK AHL BEI BEJ BEK BEL BFI BFJ BFK BFL BGI BGJ BGK BGL BHI BHJ BHK BHL

× x x ? ? x × x x x x x × × × x × × × × × x x x × ? x ? × × × ?

× x x ? x x × x x x x x × × × x × × × × × x x ? × × x ? × × × ?

× ? ? x x × x x ? x ? × × × ? × × × × x x x × × × × × × × × ×

× x x ? x x × x x ? x ? × × × x × × × × x x x × × × × × × × × ×

1H NMR control yield (%)d

89 89 100 100 76 85 84 76 76 68 66 68 81 64 60 84 52 56 58 45 64 63 56 76 81 76 77 77 81 39 69 76

single-bead construct analysise

library memberb

single-bead conventional analysisa method 1c method 2c

x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x

CEI CEJ CEK CEL CFI CFJ CFK CFL CGI CGJ CGK CGL CHI CHJ CHK CHL DEI DEJ DEK DEL DFI DFJ DFK DFL DGI DGJ DGK DGL DHI DHJ DHK DHL

× × × × × x x ? × × ? × × × × x × x x ? x x x x × x x x × × ? x

x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x

× × × × × x x ? × × x × × × × ? × ? x ? × x x ? × x x x × ? × ?

? × × × x x x ? × × × × × × ? × × × x ? x x x ? × x x × × × ? ?

× × × × x x × ? × × × × × × ? × × ? x × x x x ? × x x × × × x ×

1H NMR control yield (%)d

27 19 35 18 71 74 58 77 26 18 13 32 42 77 61 61 81 76 82 73 63 63 66 61 58 68 50 56 56 50 37 64

single-bead construct analysise x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x

x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x

a ESI-MS following single-bead conventional TFA cleavage. Each column represents one of the 4 sets of experiments. b Monomer combination (See Table 1). c See Experimental Section. d Obtained after TFA cleavage on bulk quantities of resin. e ESI-MS following single-bead analytical construct cleavage. Each column represents one of the 2 sets of experiments. x, passed (expected mass found); ×, failed; ?, indeterminate.

catch mechanism of release.26 In this particular case, 1-methylpiperazine and an isotopically labeled methylene group (Figure 2) provide the sensitizer and the peak splitter, respectively. To demonstrate the applicability of analytical construct singlebead analysis to library quality control, we assembled a model 64 member cubic (4 × 4 × 4) secondary carboxamide library on an analytical construct resin (resin 1, Figure 3). The synthesis of a one bead/one compound library was mimicked using an IRORI radio frequency system. This system, while still allowing a splitmix strategy, can provide a few milligrams (about 25 mg) of the final resin for each of the individual 64 library members. Such an amount of resin allowed for single-bead analysis, as would be conducted in a real one bead/one compound library, but it also provided enough material for bulk TFA cleavage and subsequent 1H NMR studies. The 1H NMR control experiment provides an unambiguous characterization of the library. Any reliable and informative quality control method should reflect, at the singlebead level, the results that are obtained during the 1H NMR studies. The use of the positive ESI-MS scan mode relies on the presence of protonatable functional groups in the compounds. Unfortunately, a library member does not always contain such (24) Kenner, G. W.; McDermott, J. R.; Sheppard, R. C. J. Chem. Soc., Chem. Commun. 1971, 636.

groups. To demonstrate the generality of the analytical construct approach, the model library was designed to contain, after TFA cleavage/deprotection, a broad range of functionalities. For instance, monomer B and monomer C (or L) (Table 1) give the final compound an acidic or basic functionality, respectively, but some other monomer combinations (e.g., AFJ) (Table 1) provide lipophilic neutral species after TFA cleavage. (ii) Synthesis. The solution phase synthesis of the thiopyrimidine construct linker has been described elsewhere.22 After loading this thiopyrimidine construct linker onto amino ArgoGel resin, the second step in the preparation of resin 1 (Figure 3) was the coupling of the acid labile linker to the analytical construct resin. The starting resin 1 was then distributed into 64 MicroKans, each encoded by a radio frequency ID tag (IRORI system).23,24 In the subsequent model substrate synthesis (Figure 4), reductive amination of primary amines (A, B, C, and D, first set of monomers; Table 1) provided resin-bound secondary amines. These amino groups were then acylated by the second set of monomers (4 Fmoc-protected amino acids: E, F, G, and H; Table 1). After Fmoc removal, the last set of monomers (4 carboxylic acids: I, J, K, and L; Table 1) was used to cap the resin-bound amines. Each of the microreactors contained after this final synthetic step ∼25 milligrams of a resin associated with one Analytical Chemistry, Vol. 73, No. 5, March 1, 2001

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Figure 5. Electrospray MS of the library component corresponding to monomer combination AGK belonging to the “passed” category (a) using conventional cleavage (expected mass, [M + H]+ ) 325) and (b) using analytical construct fragment release (expected mass, [M + H]+ ) 897.5)

identified member of the 4 × 4 × 4 split-mix library. After completion of the synthesis, we spatially arranged the MicroKans according to their ID tags. (2) Analysis of the 64 Library Components. (i) 1H NMR control experiment. We carried out a 1H NMR characterization of every member of the library after TFA cleavage on bulk quantities of resin removed from the MicroKans reactors. Interpretation of each of the 64 1H NMR spectra proved consistent with the expected structures. In addition, the compounds were generally obtained in good overall yield and purity (Table 2). The 1H NMR characterization of the library, thus, proved totally satisfactory, and any reliable analytical technique applied to quality control of this model library should reflect the success of the synthesis. (ii) Conventional Single Bead ESI-MS Analysis. Single beads were picked manually from each MicroKan and cleaved conventionally at linker 2 with TFA, thus providing 64 samples. These were then analyzed by mass spectrometry to follow our conventional procedure for library quality control. TFA cleavage and analysis of single beads from the whole library was performed in duplicate. The cleavage was performed following the two standard methods (Methods 1 and 2; see Experimental Section) commonly used in our laboratory for conventional single-bead analysis. Following mass spectrometry analysis in positive scan mode, the results were divided into 3 categories: the first category contained those spectra which displayed the expected mass ion for the cleaved substrate with a high signal-to-noise ratio, and the second category contained spectra showing no expected mass. The third category consisted of spectra exhibiting, in addition to the expected mass, a large number of extraneous peaks or a low signal-to-noise ratio. In this latter category, we also put compounds with mass ion detected in only one set of the 4 experiments. In terms of quality control, these 3 categories would correspond to “passed” (first category), “failed” (second category), or “indeterminate” (third category) samples, respectively. Out of 64, only 25 samples showed the expected mass (“passed” category, Figure 5a), and the expected mass was entirely 968

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absent in 28 spectra (“failed” category, Figure 6a). In the “indeterminate” category, we placed 11 spectra for which it was hard to determine whether the observed peak was an artifact or if it corresponded to the expected structure. It is noteworthy that even the 25 spectra in which we could confidently identify the appropriate ion often contain extraneous peaks in addition to the molecular ion. In addition, the need to create the third (indeterminate) category illustrates some of the problems generally encountered when undertaking single-beadlibrary analysis. The need for the operator to interpret one after another ambiguous spectra represents a bottleneck which slows down the processing of data provided by an otherwise highthroughput method. An even more important problem also appears when considering the second (failed) category: for those samples, the crucial question of whether the expected compound is present or not remains unanswered. In our example, the number of spectra showing the predicted mass is too low, and no reliable decision about using the library could be made. After such a quality control, one could mistakenly judge that the library synthesis needs to be refined, and this would require another solid-phase reaction reinvestigation to be undertaken. The importance of this strategic decision cannot be underestimated, because a library development phase is a time-consuming and expensive process. In the present case, however, the 1H NMR control experiment had unambiguously demonstrated that any of the 64 library components was present in the sample after TFA cleavage, in good yield and purity. As a result, poor ionization properties are to be invoked for those compounds which were undetectable by mass spectrometry at the single-bead concentration level. This evidences an inherent pitfall of electrospray mass spectrometry when applied to single-bead characterization: even a totally successful library synthesis may reveal only a fraction of its components (in our case, 40% for this 100% successful library synthesis). This type of analysis protocol, thus, appears relatively uninformative and misleading.

Figure 6. Electrospray MS of the library component corresponding to monomer combination BGI belonging to the “failed” category (a) using conventional cleavage (expected mass: [M + H]+ ) 296) and (b) using analytical construct fragment release (expected mass, [M + H]+ ) 924.5).

Figure 7. Electrospray MS of the library component corresponding to monomer combination BFK belonging to the “indeterminate” category (a) using conventional cleavage (expected mass: [M + H]+ ) 341) and (b) using analytical construct fragment release (expected mass, [M + H]+ ) 969.5).

(iii) Single Bead ESI-MS Analysis Using Analytical Construct Methodology. Oxidative activation and nucleophilic substitution on single beads picked from the MicroKans (Figure 2, route a) provided the 64 analytical construct fragments (experiment performed in duplicate). In contrast to conventional analysis, the spectra obtained are all straightforward to interpret. An easily identified peak split doublet corresponding to the expected molecular ion is by far the main signal in each spectrum, and the signal-to-noise ratio is gratifyingly high (Figures 5b, 6b, 7b). The amine functionality on the construct sensitized the entire fragment to ESI-MS in positive scan mode and rendered all substrates, without exception, now readily visible. After a library analysis conducted in this fashion, one can draw the correct conclusion that the library synthesis proceeded well, as verified from the 1H NMR control experiment described above. As a consequence, the

analytical construct-based library evaluation appears to be much more informative and reliable in comparison to conventional quality control, the analytical technique unmasking the identity of the actual species present on the bead. In all cases, each 64single-bead analysis and its duplicate gave an equally good and reproducible spectrum. The comparison of the spectra obtained after construct cleavage with the spectra obtained after TFA cleavage illustrates the value of the analytical construct methodology. For instance, although in spectrum 7a the expected mass was found, the presence of other peaks with similar intensity was confusing, and no clear assumption could be drawn. The interpretation becomes straightforward when the analytical construct methodology is employed, as spectrum 7b shows with a unique doublet at the expected mass. However, the potency of our new analytical Analytical Chemistry, Vol. 73, No. 5, March 1, 2001

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approach appears even more obvious when comparing spectrum 6b to 6a: an intense signal at the expected mass is now clearly visible, and no signal at all could be obtained by electrospray mass spectrometry technique used in a conventional way. Moreover, the comparative data in Table 2 clearly show a main asset of this methodology. Analytical constructs not only improved the spectrum quality in the individual cases that were previously described, but above all, guarantee good quality spectra for any of the 64 samples, even when they display a broad range of functional groups. CONCLUSIONS We have successfully demonstrated that the analytical construct approach facilitates single-bead analysis and, as a consequence, allows for quality control of split-mix libraries. Cleavage at the analytical construct linker rendered all of these compounds, without exception, visible by electrospray mass spectrometry. In addition, all of the construct related compounds were peak-split, thus uniquely identified. Conversely, after conventional TFA cleavage at linker 2, we found that a large number of compounds were invisible to ESI-MS analysis that was conducted in a conventional way. In view of the excellent results obtained in this proof of concept study, we believe that by incorporating analytical construct methodology into our libraries, we have made an important step toward solving current quality control problems. As emphasized above, ESI-MS provides a unique potential for high-throughput analysis and robotic operation. As a result, automated systems have been developed in pharmaceutical

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industries to cope with the measurement of thousands of combinatorial chemistry samples per month. Because of the clarity of the spectra we obtained using the analytical construct methodology, we think that operator judgments during quality control could be supported and, therefore, speeded up by softwares designed to select only doublets separated by a peak split of 2. The presence of byproducts could also be signaled, providing valuable data for the optimization of reaction conditions or interpretation of screening results. Abbreviations: HATU, azabenzotriazolyl-N,N,N′,N′-tetramethyluronium hexafluorophosphate; DIPEA, diisopropylethylamine; DMF, dimethylformamide; DCM, dichloromethane; Et2O, diethyl ether; TFA, trifluoroacetic acid; PyBOP, benzotriazole-1-yl-oxytris-pyrrolidino-phosphonium hexafluorophosphate; Fmoc, 9-fluorenylmethoxycarbonyl; DMSO, dimethyl sulfoxide. ACKNOWLEDGMENT We thank Yih-Sang Pang for his assistance with electrospray MS.

Received for review July 17, 2000. Accepted December 7, 2000. AC000814Y (25) Mckeown, S. C.; Watson, S. P.; Carr, R. A. E.; Marshall, P. Tetrahedron Lett. 1999, 40, 2407-2410. (26) Geysen, H. M.; Wagner, C. D.; Bodnar, W. M.; Markworth, C. J.; Parke, G. J.; Schoenen, F. J.; Wagner, D. S.; Kinder, D. S. Chem. Biol. 1996, 3, 679.