Swartz Analytical Techniques in Combinatorial Chemistry

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The Use of Mass Spectrometry

Annette Hauser-Fang and Paul Vou´ros

Northeastern University

Boston, Massachusetts

Combinatorial chemistry has evolved as a major area of interest for the pharmaceutical industry and has created a need for methods of characterization that are distinctly different from the classical tools of analytical organic chemistry like nuclear magnetic resonance (NMR) and C, H, and N analysis (1,2). There are currently several approaches for combinatorial chemistry that differ from each other in many ways (3–14). One can generate large solvated mixtures of compounds with as many as 106 different components in one sample, the so-called true libraries (15), or one can use the microtiter plate approach that is used to synthesize similar numbers of compounds individually on microtiter plates within small wells, so that each well contains only one or very few components. Clearly, for the second approach, synthesis and analysis must be automated to be efficient. Alternatively, many combinatorial libraries have been synthesized on solid supports (16–20) using polymeric beads. Advantages for solid phase chemistry include more efficient and simplified sample cleanup because washing of the beads usually removes all excess reagents and byproducts. This is especially true for the so-called mix-and-split synthesis (21) where each bead carries one component and which has been used with success by synthetic chemists. Single-bead analysis has been a good match for matrixassisted laser desorption ionization (MALDI) and several publications have shown that this approach can provide excellent data as is shown below.

Within the context of these new developments in drug discovery, mass

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spectrometry (MS) has begun to assume a leading role (22,23) that, in addition to the traditional analysis of synthetic samples, appears to enter into the examination of molecular recognition and screening for lead drug candidates. This chapter focuses on a review of the use of MS techniques for the characterization and the biological screening of combinatorial libraries, often in combination with on-line separation methods.

I.CHARACTERIZATION OF COMBINATORIAL LIBRARIES BY MASS SPECTROMETRY

With the development of combinatorial chemistry it has become necessary to develop methods of analysis that are applicable to the characterization of components out of mixtures of molecules. It is often not sufficient to rely on biological screening procedures and later identification of active compounds, but it may also be necessary to identify synthetic products and verify the generation of a certain diversity within the combinatorial library. Establishing the integrity of a library can be important in order to avoid false positives and/ or false negatives that may result from the presence of impurities (24) or the absence of products anticipated in the synthesis. When opting for certain approaches, the overall effort, the cost of purchasing and maintaining expensive equipment, and the superior contribution of certain methods toward the analysis have to be considered. The following sections give an overview of the applicability of some of the most popular techniques.

A.Low-Resolution CE-MS AND CE-MS/MS Methods Using Triple Quadrupole Mass Spectrometers or Quadrupole Ion Traps

A number of publications have focused on the characterization of combinatorial libraries by direct infusion, liquid chromatography (LC), or capillary electrophoresis (CE) (25–29) coupled to electrospray ionization-mass spectrometry (ESI-MS), electrospray ionization-tandem mass spectrometry (ESI-MS/ MS) (30–37), nanoelectrospray-MS (38,39), and atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) (40–42) using triple quadrupole instruments. Results obtained by direct infusion of a library into an ESI-MS system may be fraught with uncertainty even when dealing with small libraries because of the low resolving power of the triple-quadrupole instrument. It is not uncommon to find peaks at virtually every single mass unit depending on the library size. In fact, even relatively small libraries with, for example, 30 components give rise to at least 60 peaks due to isotopes. Counting in a few

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impurities one can easily confuse the spectrum and generate many false positives. Moreover, the ionization efficiencies of individual library components may vary widely whether in the positive or negative ionization mode. Establishing a ‘‘hit’’ from a given ion mass signal may thus be a questionable proposition and even more questionable can be the assessment of molecular ratios of library constituents. Accordingly, determination of ionization efficiencies of library members is necessary before the presence of nonisobaric compounds can be fully established. The combined use of positive and negative ion detection may often provide considerable flexibility in the characterization of diverse libraries. Figure 1 shows an example by Dunayevskiy et al. (27,30) where switching between positive and negative ionization can help distinguish real library components from impurities. Ideally, under low resolution MS conditions prior to separation of the library mixture by CE or LC is advisable along with the need to aquire MS/MS spectra of every peak. But even CE-MS and LC-MS only help separate a mixture for the identification of isobaric components. They do not provide a fingerprint of a compound and, unless the retention time of the compound of interest is known, it is still possible that an isobaric impurity has been identified as a library component. In low-resolution MS, fragmentation spectra are always the most definitive proof of a compound’s existence. Figure 2 shows an application where the three dimensional presentation of LC-MS and LC-MS/MS data is used to detect structural trends within the eluted peaks, therefore permitting, for example, the easy identification of library components belonging to certain subgroups (43). On the other hand, it is frequently desirable to only determine the reproducibility of the synthesis of a specific library. In such a case, the ESI-MS profile may be deemed sufficient for general library characterization.

The new development of the quadrupole ion trap technology with its possibility of automated recognition of peaks and subsequent fragmentation using MSn has simplified the process of fingerprinting of components by fragmentation. It is possible to get completely automated MSn information from every HPLC peak just on the basis of programming the instrument to fragment the nth most intense ion in the spectrum until the MSn step. This is helpful for unattended analysis runs (e.g., overnight) and improves the chances of complete identification of components from their fragmentation spectra (44).

Theoretical and experimental results by Blom (45) have focused on the precision requirements for the mass spectrometric analysis of combinatorial library mixtures. In his work, Blom has used discrete mass filters in combination with mass-MS/MS, M 1/M, and M 2/M isotope ratio filters to determine library components specifically from peptide libraries. As pointed out below, while nonpeptide libraries have mass distributions that are completely random, peptide library building blocks are limited in their diversity,

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Figure 1 Mass spectrometric analysis of combinatorial libraries L1–L6: gray, detected in positive ion ESI; black, detected in negative ion ESI; vertical stripes, detected in both modes; X, detected in MS/MS experiment. (Reprinted from Ref. 30.)

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Figure 2 (Top) 3-D surface rendering of a full scan LC-MS dataset obtained on a 625-combinatorial-peptide library. (Middle) 3-D extracted ion surface of m/z 496, indicating three major regions. (Bottom) 3-D software neutral loss (NL-264) surface, indicating components that contain substructure V-F-COOH. (Reprinted from Ref. 43.)

and this leads to clustering of the library components around certain m/z values. The libraries used in this example were complex and in the best case only 6% of their components could be characterized by their integer masses. Use of a mass filter alone clearly produced too poor a fingerprint for unambiguous characterization of all components. Therefore all available mass spectrometric filters were made to work in a complementary fashion in order to recognize impurities and distinguish peptide components from one another. For the

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experimental part of the work by Blom, a software-modified triple-quadrupole mass spectrometer was employed that allowed automated switching between MS and MS/MS modes whenever the peak intensity of an expected candidate peak was of such a level as to trigger fragmentation.

Automation (37,41,42,46–53) has not only enabled researchers to analyze their library components more efficiently but has also helped in the synthetic process. Many companies (50,54) have adopted the approach of synthesizing one or two components of a library separately in a small well using microtiter plates as reaction vessels and robots for the addition of reagents and mixing. The microtiter plates with the individual library components are then automatically analyzed by low resolution MS (54) or high-resolution Fourier transform ion cyclotron-mass spectrometry (FTICR-MS) (55). Most setups include HPLC equipment with an autosampler to do continuous flow injections and molecular weight analysis of the contents of each well on the microtiter plate. No MS/MS is needed because one mass filter is usually enough for the confirmation of the one expected molecular weight and maybe a few impurity peaks. Samples can be analyzed overnight for maximum efficiency. A typical setup is shown in Fig. 3 (56). However, it should be pointed out that the absence of additional peaks in the mass spectrum does not guarantee 100% sample purity in the titer well. The possibility of false negatives always needs to be addressed by an alternative method of detection because some components (or impurities) may be transparent to ESI ionization.

Figure 3 High-throughput QA schematic. (Reprinted from Ref. 56.)

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B.Matrix-Assisted Laser Desorption Ionization and Other Desorption Ionization Techniques for the Identification of Combinatorial Libraries

There have been many publications focusing on matrix assisted laser desorption ionization (MALDI) (49,57–59, 60–72), secondary ion mass spectrometry (SIMS) (73–76), and 252Cf Plasma desorption mass spectrometry (77–79) as a tool for the analysis of combinatorial libraries using different kinds of mass spectrometers. MALDI is generally not an ionization technique that can be easily coupled to any separation method but, since many libraries today consist of physically separated compounds on beads or within wells on microtiter plates, separation is often not necessary. It is important though, to understand the limitations and advantages of the MALDI approach because only libraries with relatively high molecular weight components (m/z 500) are suitable for analysis due to matrix interferences at the lower mass ranges in the spectrum. That may be one of the reasons why a significant part of the research using MALDI has focused on the analysis of peptide libraries (58– 63, 80–83). MALDI has been used with both time-of-flight (TOF) (65,75) and Fourier transform ion cyclotron resonance mass spectrometry (49, 84) for the analysis of split pool and encoded combinatorial libraries (66–68,70,85,86).

It has been shown by Egner et al. (64,65) that it is possible to analyze compounds directly from prepared polystyrene beads. This approach has been useful to verify and improve the synthetic method for the generation of combinatorial libraries. Assuming that there are approximately 106 beads in 1 gram of polystyrene resin, and the substitution factor for the solid phase chemistry is 0.4 mmol/g or more, there is about 400 pmol of compound attached to each single bead. MALDI-TOF analysis has detection limits in the femtomole region, which makes it compatible with single-bead analysis (76).

In preparation for the experiments, the library components were removed from the bead by exposing it to trifluoroacetic acid vapor for 30 min. After the cleavage reaction was complete an internal standard was added together with the matrix solution for the laser desorption. The matrix was formed around the bead within 15–30 min and MALDI-TOF analysis was performed directly from the sample well. Results are shown in Fig. 4 where a variety of peptides was monitored in addition to bradykinin, which acted as the internal calibrant. As for all MALDI experiments, it was critical that the right matrix be chosen for the analysis. After initial examinations of different matrices under a stereo microscope followed by the MALDI-TOF experiment, dihy- droxy-benzoic acid (DHP) was found to produce the largest crystals and the best results.

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Figure 4 (a) Analysis of dansyl-Ile-Thr(O But)-Pro-Gln-Trp-Lys(Boc)-Wang- linker-resin, giving peptide dansyl-Ile-Thr-Pro-Gln-Trp-Lys (MH , 1006.0). The peak at 772.6 represents a small amount of undansylated material. (b) Analysis of Fmoc- Cys(Trt)-Lys(Boc)-Ile-HMPB-linker-resin, giving Fmoc-Cys-Lys-Ile (MH , 585.5), Fmoc-Cys(Trt)-Lys-Ile (MH , 849.8), and Fmoc-Cys-Lys-Ile disulfide [(M-S-S- M)H , 1168.3]. (c)Analysis of the disulfide of Fmoc-Cys-Asn-Cys-Lys(Boc)-Ile- HMPB-linker-resin giving the disulfide of Fmoc-Cys-Asn-Cys-Lys-Ile (MH , 801.0). (Reprinted from Ref. 64.)

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Youngquist et al. (62,63) went one step further with their resin bead analysis by not only getting molecular weight information on their peptide based library components but sequencing information as well in the same experiment. Their strategy employed capping reagents at each step of the library synthesis so that beads would carry not only the library component but also a small percentage of peptides of different chain lengths. Sequencing information was obtained from the mass differences between termination products. Figure 5 shows the principle of the generation of the peptide libraries and the use of the capping reagents at every step. Termination products ideally differed by one amino acid (87).

Figure 5 Termination synthesis method to produce sequence-specific families of peptides on each resin bead in the library. After screening, active peptides are isolated and the peptide products are released from the bead (—●) by CNBr digestion. (Reprinted from Ref. 62.)

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Other important factors for a successful analysis have included the use of a linker between the bead and the synthesized peptides. This ensured efficient release from the bead and increased the molecular weight of the analytes to m/z values greater than 500, so that there was no interference with the low molecular weight noise that was produced by the desorption of the MALDI matrix. With the linker in place, the method has been sensitive enough to use only 1% of the material from an 88- m bead for sequencing. Decreasing the bead diameter makes automated library sorting possible. Results were obtained from beads as small as 17 m in diameter. The sequencing speed that was obtained using this method was 25–30 residues per hour and 77 of 80 beads were sequenced successfully with only one instance where the order of two amino acids could not be determined.

For both of the above examples it was important that the method could identify byproducts. When the reaction did not go to completion, so called deletion peptides were generated that were identified from their mass differ-

Figure 6 MALDI mass spectrum of an unknown peptide. A 5% portion of the products isolated from a single bead was analyzed. (Reprinted from Ref. 62.)