combinatorial chemistry, part b

Preface Combinatorial chemistry has matured from a field where efforts initially focused on peptide-based research to become an indispensable research tool for molecular recognition, chemical-property optimization, and drug discovery. Originally used as a method to primarily generate large numbers of molecules, combinatorial chemistry has been significantly influenced and integrated with other important fields such as medicinal chemistry, analytical chemistry, syn- thetic chemistry, robotics, and computational chemistry. Even though the initial focus of attention was providing larger numbers of molecules with a ‘‘diversity’’ goal in mind, other factors came into play depending upon the problem scientists were trying to solve, such as bioactivity, solubility, permeability properties, PK, ADME, toxicity, and patentability. One can think of combinatorial chemistry and compound screening as an iterative Darwinian process of divergence and selection. Particularly in drug discovery, where time is a critical factor to success, combinatorial chemistry offers the means to test more molecule hypotheses in parallel. We will always be limited to a finite number of molecules that we can economically synthesize and evaluate. Even with all the advances in automa- tion technologies, combinatorial chemistry, and higher-throughput screens that improve our ability to rapidly confirm or disprove hypotheses, the synthesis and screening cycle remains the rate-determining process. Fortunately, we continue to make great strides forward in the quality and refinement of pre- dictive algorithms and in the breadth of the training sets amassed to aid in the drug discovery/compound optimization iterative process. Anyone who has optimized chemical reactions for combinatorial libraries or process chemistry knows first hand how much experimentation is required to identify optimal conditions. Chemical feasibility is at the heart of small mol- ecule discovery and chemotype prioritization since it essentially defines what can and cannot be analoged (i.e., analogability). Although analogability is not the only driving factor, quite often it is overlooked. For example, when com- mercially-available compounds or complex natural products are screened, the leads generated are often dropped because of the difficulty to rapidly analog them in the lead optimization stage. The desirability of a chemotype is a function of drug-likeness, potency, novelty, and analogability. A particularly attractive feature of combinatorial chemistry is that when desirable properties are identified, they can often be xiii optimized through second-generation libraries following optimized synthetic protocols. If this process of exploring truly synthetically accessible chemical spaces could be automated, then it would open up the exciting possibility of modeling the iterative synthesis and screening cycle. Predicting, or even just mapping, synthetic feasibility is a sleeping giant; few people are looking into it, and the ramifications of a breakthrough would be revolutionary for both chemistry and drug discovery. In-roads to predicting (or even just mapping) chemical feasibility have the potential to have as large an impact on drug discovery as computational models of bioavailability and drugability. These are important questions where scientists are now starting to generate a large-enough body of information on high-throughput synthetic chemistry to begin to more globally understand what is cost-effectively pos- sible. Within the biopharmaceutical industry, significant investments in new technologies have been made in molecular biology, genomics, and proteomics. However, with the exception of combinatorial chemistry, relatively little has been done to advance the fundamental nature of chemistry in drug discovery from a conceptual perspective. Now, after having gone through the molecule-generating period where research institutions have a large historical compound collection and the pro- liferation of combinatorial chemistry services, the trend is now after making more targeted-oriented molecular entities also known as ‘‘focused libraries.’’ An important emerging question is: How can one most effectively make the best possible ‘‘focused libraries’’ to answer very specific research questions, given all the possible molecules one could theoretically synthesize? The first installment in this series (Volume 267, 1996) mostly covered peptide and peptidomimetic based research with just a few examples of small molecule libraries. In this volume we have compiled cutting-edge research in combinatorial chemistry, including divergent areas such as novel analytical techniques, microwave-assisted synthesis, novel linkers, and synthetic ap- proaches in both solid-phase and polymer-assisted synthesis of peptides, small molecules, and heterocyclic systems, as well as the application of these tech- nologies to optimize molecular properties of scientific and commercial interest. Guillermo A. Morales Barry A. Bunin xiv preface METHODS IN ENZYMOLOGY EDITORS-IN-CHIEF John N. Abelson Melvin I. Simon DIVISION OF BIOLOGY CALIFORNIA INSTITUTE OF TECHNOLOGY PASADENA, CALIFORNIA FOUNDING EDITORS Sidney P. Colowick and Nathan O. Kaplan Contributors to Volume 369 Article numbers are in parentheses and following the names of contributors. Affiliations listed are current. Fernando Albericio (2), University of Barcelona, Barcelona Biomedical Research Institute, Barcelona Science Park, Josep Samitier 1, Barcelona, 08028, Spain Alessandra Bartolozzi (19), Surface Logix, Inc., 50 Soldiers Field Place, Brighton, Massachusetts, 02135 Hugues Bienayme ´ (24), Chrysalon Mo- lecular Research, IRC, 11 Albert Einstein Avenue, Villeurbannem, 69100, France Sylvie E. Blondelle (18), Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California, 92121 Ce ´ sar Boggiano (18), Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California, 92121 Stefan Bra ¨ se (7), Institut fu ¨ r Organische Chemie, Universita ¨ t Karlsruhe (TH), Fritz-Haber-Weg 6, Karlsruhe, D-76131, Germany Andrew M. Bray (3), Mimotopes Pty Ltd., 11 Duerdin Street, Clayton, Vic- toria, 3168, Australia Wolfgang K D. Brill (23), Discovery Research Oncology, Pharmacia Italy S.p.A, Viale Pasteur 10, Nerviano (MI), I-20014, Italy Max Broadhurst (14), Alchemia Pty Ltd., Eight Mile Plains, Queensland 4113, Aus- tralia Balan Chenera (24), Amgen Inc., Depart- ment of Small Molecule Drug Discovery, One Amgen Center Drive, Thousand Oaks, California, 91320 James W. Christensen (5), Advanced ChemTech Inc., 5609 Fern Valley Road, Louisville, Kentucky, 40228 Andrew P. Combs (12), Incyte Corpo- ration,Wilmington, Delaware, 19880-0500 Scott M. Cowell (16), Department of Chemistry, University of Arizona, Tucson, Arizona, 85721 Stefan Dahmen (7), Institut fur Orga- nische Chemie, RWTH Aachen, Pirlet- Str. 1, Aachen, 52074, Germany Ninh Doan (17), Division of Hematology and Oncology, Department of Internal Medicine, UC Davis Cancer Center, Uni- versity of California Davis, Sacramento, California, 95817 Roland E. Dolle (8), Senior Director of Chemistry, Department of Chemistry, Adolor Corporation, 700 Pennsylvania Drive, Exton, Pennsylvania, 19345 Nicholas Drinnan (14), Alchemia Pty Ltd., Eight Mile Plains, Queensland 4113, Australia Amanda M. Enstrom (17), Division of Hematology and Oncology, Department of Internal Medicine, UC Davis Cancer Center, University of California Davis, Sacramento, California, 95817 ix Liling Fang (1), ChemRx Division, Dis- covery Partners International, 385 Oyster Point Boulevard, Suite 1, South San Francisco, California, 94080 Eduard R. Felder (23), Discovery Re- search Oncology, Pharmacia Italy S.p.A., Viale Pasteur 10, Nerviano (MI), I-20014, Italy A ´ rpa ´ d Furka (5), Eo ¨ tvo ¨ s Lora ´ nd Univer- sity, Department of Organic Chemistry, P.O. Box 32, Budapest 112, H-1518, Hungary A. Ganesan (22), University of Southamp- ton, Department of Chemistry, Highfield, Southampton, SO17 1BJ, United Kingdom J. Gabriel Garcia (20), 4SC AG, Am Klopferspitz 19A, 82152, Martinsried, Germany Brian Glass (13), Incyte Corporation, Wilmington, Delaware, 19880-0500 Matthias Grathwohl (14), Alchemia Pty Ltd., Eight Mile Plains, Queenland 4113, Australia Michael J. Grogan (19), Surface Logix, Inc., 50 Soldiers Field Place, Brighton, Massachusetts, 02135 Xuyuan Gu (16), Department of Chemistry, University of Arizona, Tuscon, Arizona, 85721 Eric Healy (5), Advanced ChemTech Inc., 5609 Fern Valley Road, Louisville, Kentucky, 40228 Timothy F. Herpin (4), Rho ˆ ne-Poulenec Rorer, 500 Arcola Road, Collegeville, Pennsylvania, 19426 Cornelia E. Hoesl (25), Torrey Pines In- stitute, Room 2-136, 3550 General Atom- ics Court, San Diego, California, 92121 Christopher P. Holmes (9), Affymax Inc., 4001 Miranda Avenue, Palo Alto, California, 94304 Richard Houghten (25), Torrey Pines In- stitute for Molecular Studies, 3550 Gen- eral Atomics Court, Room 2-136, San Diego, California, 92121 Victor J. Hruby (16), Department of Chemistry, University of Arizona, Tucson, Arizona, 85721 Christopher Hulme (24), Amgen Inc., De- partment of Small Molecule Drug Discov- ery, One Amgen Center Drive, 29-1-B, Thousand Oaks, California, 91320 Sharon A. Jackson (12), Aventis Pharma- ceuticals, 202-206, Bridgewater, New Jersey, 08807-0800 Ian W. James (3), Mimotopes Pty Ltd., 11 Duerdin Street, Clayton, Victoria, 3168, Australia Wyeth Jones (24), Amgen Inc., Depart- ment of Small Molecule Drug Discovery, One Amgen Center Drive, 29-1-B, Thou- sand Oaks, California, 91320 Patrick Jouin (10), CNRS UPR 9023, CCIPE, 141, rue de la Cardonille, Mont- pellier Cedex 05, 34094, France C. Oliver Kappe (11), Institute of Chemis- try, Karl-Franzens-University Graz, Heinrichstrasse 28, Graz, A-8010, Austria Steven A. Kates (19), Surface Logix, Inc., 50 Soldiers Field Place, Brighton, Massa- chusetts, 02135 Viktor Krchn ˇ a ´ k (6), Torviq, 3251 West Lambert Lane, Tuscon, Arizona, 85742 Kit S. Lam (15, 17), Division of Hematol- ogy and Oncology, Department of In- ternal Medicine, UC Davis Cancer Center, University of California Davis, Sacramento, California, 95817 Alan L. Lehman (17), Division of Hema- tology and Oncology, Department of In- ternal Medicine, UC Davis Cancer Center, University of California Davis, Sacramento, California, 95817 x contributors to volume 369 Ruiwu Liu (15, 17), Division of Hematol- ogy and Oncology, Department of In- ternal Medicine, UC Davis Cancer Center, University of California Davis, Sacramento, California, 95817 Matthias Lormann (7), Kekule ´ -Institut fu ¨ r Organische Chemie und Biochemie der Rheinischen, Friedrich Wilhelms Univer- sita ¨ t Bonn, Gerhard-Domagk-Strasse 1, Bonn, D-53121, Germany Jan Marik (15), Division of Hematology and Oncology, Department of Internal Medicine, UC Davis Cancer Center, Uni- versity of California Davis, Sacramento, California, 95817 Katia Martina (23), Discovery Research Oncology, Pharmacia Italy S.p.A., Viale Pasteur 10, Nerviano (MI), I-20014, Italy Joeseph Maxwell (17), Division of Hema- tology and Oncology, Department of In- ternal Medicine, UC Davis Cancer Center, University of California Davis, Sacramento, California, 95817 Wim Meutermans (14), Alchemia Pty Ltd., 3 Hi-Tech Court, Brisbane Technology Park, Eight Mile Plains, QLD 4113, Aus- tralia George C. Morton (4), Rho ˆ ne-Poulenc Rorer, 500 Arcola Road, Collegeville, Pennsylvania, 19426 Adel Nefzi (25), Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California, 92121 Thomas Nixey (24), Amgen Inc., Depart- ment of Small Molecule Drug Discovery, One Amgen Center Drive, 29-1-B, Thou- sand Oaks, California, 91320 John M. Ostresh (25), Torrey Pines Insti- tute, Room 2-136, 3550 General Atomics Court, San Diego, California 92121 Vitecek Pade ˇ ra (6), Torvic, 3251 W Lam- bert Lane, Tucson, Arizona, 84742 E.R. Palmacci (13), 77 Massachusetts Avenue, T18-209, Cambridge, Massachu- setts, 02139 Yijun Pan (9), Affymax Inc., 4001 Mi- randa Avenue, Palo Alto, California, 94304 Jack G. Parsons (3), Mimotopes Pty Ltd., 11 Duerdin Street, Clayton, Victoria, 3168, Australia Robert Pascal (10), UMR 5073, Univer- site ´ de Montpellier 2, CC017, place Euge ` ne Bataillon, Montpellier Cedex 05, F-34094, France Clemencia Pinilla (18), Torrey Pines In- stitute for Molecular Studies and Mixture Sciences, Inc., 3550 General Atomics Court, San Diego, California, 92121 Obadiah J. Plante (13), Massachusetts Institute of Technology, Department of Chemistry, 77 Massachusetts Avenue, Cambridge, Massachusetts, 02139-4307 Gregory Qushair (2), University of Barcelona, Barcelona Biomedical Research Institute, Barcelona Science Park, Josep Samitier 1, Barcelona, 08028, Spain Jorg Rademann (21), Eberhard-Karls-Uni- versity, Tu ¨ bingen, Institute of Organic Chemistry, Auf der Morgenstelle 18, Tu ¨ - bingen, 72076, Germany Joseph M. Salvino (8), Director of Com- binational Chemistry, Adolor Corpor- ation, 700 Pennsylvania Drive, Exton, Pennsylvania, 19345 Peter H. Seeberger (13), Laboratorium fuer Organische Chemie, HCI F 315, Wolfgang-Pauli-Str. 10, ETH-Hoengger- berg, CH-8093 Zu ¨ rich, Switzerland Craig S. Sheehan (3), Mimotopes Pty Ltd., 11 Duerdin Street, Clayton, Vic- toria, 3168, Australia contributors to volume 369 xi Adrian L. Smith (24), Amgen Inc., Depart- ment of Small Molecule Drug Discovery, One Amgen Center Drive, Thousand Oaks, California, 91320 Re ´ gine Sola (10), UMR 5076, Ecole Nationale Supe ´ rieure de Chimie de Montpellier, 8, rue Delaware l’Ecole Normale, Montpellier Cedex 05, F- 34296, France Aimin Song (17), University of California, UC Davis Cancer Center, Division of Hematology and Oncology, 4501 X Street, Sacramento, California, 95817 Alexander Stadler (11), Institute of Chemistry, Karl-Franzens-University Graz, Heinrichstrasse 28, Graz, A-8010, Austria Paul Tempest (24), Amgen Inc., Depart- ment of Small Molecule Drug Discovery, One Amgen Center Drive, 29-1-B, Thou- sand Oaks, California, 91320 David Tumelty (9), Affymax, Inc., 4001 Miranda Avenue, Palo Alto, California, 94304 Josef Vagner (16), Department of Chem- istry, University of Arizona, Tuscon, Ari- zona, 85741 Jesus Vazquez (2), University of Barce- lona, Barcelona Biomedical Research Institute, Barcelona Science Park, Josep Samitier 1, Barcelona, 08028, Spain Michael L. West (14), Alchemia Pty Ltd., Eight Mile Plains, Queensland 4113, Australia Zemin Wu (3), Mimotopes Pty Ltd., 11 Duerdin Street, Clayton, Victoria, 3168, Australia Bing Yan (1), ChemRx Division, Discovery Partners International, 385 Oyster Point, Boulevard, Suite 1, South San Francisco, California, 94080 Yongping Yu (25), Torrey Pines Institute, Room 2-136, 3550 General Atomics Court, San Diego, California, 92121 Florencio Zaragoza (26), Medicinal Chemistry, Novo Nordisk A/S, Novo Nor- disk Park, Malov, 2760, Denmark Jiang Zhao (1), ChemRx Division, Discov- ery Partners International, 385 Oyster Point Boulevard, Suite 1, South San Francisco, California, 94080 xii contributors to volume 369 [1] High-Throughput Parallel LC/UV/MS Analysis of Combinatorial Libraries By Liling Fang,Jiang Zhao,andBing Yan Introduction Combinatorial chemistry and high-throughput organic synthesis allow the preparation of a large number of diverse compounds in a relative short period of time in order to accelerate discovery efforts in the pharmaceut- ical and other industries. A library can comprise hundreds to thousands of compounds with the need to rapidly analyze those compounds for their identity and purity. Different compound separation and mass spectrometry (MS) techniques have been applied for the characterization of combinator- ial libraries. These include separation techniques such as liquid chromatog- raphy (LC) and capillary electrophoresis and different ionization methods and mass analyzers. 1–3 LC/MS * is the most popular technique used in com- binatorial library analysis because it combines separation, molecular weight determination, and relative purity evaluation in a single sample in- jection. However, the throughput of conventional LC/MS could not meet the need to analyze every member in a large combinatorial library in a timely fashion. Higher-throughput analysis was achieved by utilizing shorter columns at higher flow rates. 4 Supercritical fluid chromatography (SFC)/MS has 1 A. Hauser-Fang and P. Vouros, ‘‘Analytical Techniques in Combinatorial Chemistry’’ (M. E. Swartz, ed.). Marcel Dekker, New York, 2000. 2 B. Yan, ‘‘Analytical Methods in Combinatorial Chemistry.’’ Technomic, Lancaster, 2000. 3 D. G. Schmid, P. Grosche, H. Bandel, and G. Jung, Biotechnol. Bioeng. Comb. Chem. 71, 149 (2001). *Abbreviations: CLND, chemiluminescence nitrogen detection; C log P, calculated partition coefficient; ELSD, evaporative light scattering detection; ESI-MS, electrospray ionization mass spectrometry; FWHM, full width at half maximum; i.d., inner diameter; LC, HPLC, liquid chromatography, high-performance liquid chromatography; LC/MS, liquid chroma- tography – mass spectrometry; LC/MS/MS, liquid chromatography – mass spectrometry – mass spectrometry; LC/UV/MS, liquid chromatography mass spectrometry with a UV detector; LIB, compound library; log P, water/octanol partition coefficient; MUX, multiplexed; RSD, relative standard deviation; SFC, supercritical fluid chromatography; TFA, trifluoroacetic acid; TIC, total ion current; TOF, time of flight; TOFMS, time of flight mass spectrometry. 4 H. Lee, L. Li, and J. Kyranos, Proceedings of the 47th ASMS Conference on Mass Spectrometry and Allied Topics, Dallas, Texas, June 13–17, 1999. [1] high-throughput LC/UV/MS analysis of libraries 3 Copyright 2003, Elsevier Inc. All rights reserved. METHODS IN ENZYMOLOGY, VOL. 369 0076-6879/03 $35.00 been used to achieve desirable high speed taking advantage of the low vis- cosity of CO 2 . 5 However, the serial LC/MS approach by its nature does not match the speed of parallel synthesis. Parallel LC/MS is the method of choice to increase throughput while maintaining the separation efficiency. An eight-probe Gilson 215/889 autosampler was incorporated into a quadruple mass spectrometer. 6 This arrangement enabled the injection of eight samples (a column from a 96-well microtiter plate) simultaneously for flow-injection analysis/MS (FIA-MS) analysis to achieve a throughput of 8 samples/min. A novel multiplexed electrospray interface (MUX) 7 was developed in 1999 and became commercially available for parallel high-throughput LC/UV/MS analysis. The eight-way MUX consists of eight nebulization-assisted electrospray ionization sprayers, a desolvation gas heater probe, and a rotating aperture. It can accommodate all eight high-performance liquid chromatograph (HPLC) streams at a reduced flow rate of <100 l/min per stream and conduct electrospray ionization for all eight streams simultaneously. Ions are continuously formed at the tip of each sprayer and the MUX interface allows sprayers to be sampled sequen- tially using the rotating aperture driven by a programmable stepper motor. At any given time, only ions from one stream are admitted into the ion sampling cone, while ions from the other seven sprayers are shielded. Each liquid stream is sampled for a preset time with mass spectra acquired in full mass range into eight simultaneously open data files synchronized with the spray being sampled. With a 0.1-s acquisition time per sprayer and 0.05-s intersprayer delay time, the time-of-flight (TOF) mass analyzer can acquire a discrete data file of electrospray ion current sampled from each stream over the entire HPLC separation with a cycle time of 1.2 s. Therefore, this eight-way MUX-LCT was like having eight individual electrospray ionization (ESI)-MS systems working simultaneously. The MUX interface enables the coupling of parallel liquid chromatog- raphy to a single mass spectrometer. This technology has had a great impact in high-throughput LC/MS analysis. In drug development, a four- way MUX interface was used on a triple quadrupole mass spectrometer to simultaneously validate LC/MS/MS methods for the quantitation of loratadine and its metabolite in four different biological matrixes 8 and of 5 M. C. Ventura, W. P. Farrell, C. M. Aurigemma, and M. J. Greig, Anal. Chem. 71, 2410 (1999). 6 T. Wang, L. Zeng, T. Strader, L. Burton, and D. B. Kassel, Rapid Commun. Mass Spectrom. 12, 1123 (1998). 7 V. De Biasil, N. Haskins, A. Organ, R. Bateman, K. Giles, and S. Jarvis, Rapid Commun. Mass Spectrom. 13, 1165 (1999). 8 M. K. Bayliss, D. Little, D. M. Mallett, and R. S. Plumb, Rapid Commun. Mass Spectrom. 14, 2039 (2000). 4 analytical techniques [1] diazepam in rat liver microsomes for in vitro metabolic stability. 9 The four- channel LC/MS/MS system was also reported for the quantification of a drug in plasma on both the narrow-bore and capillary scales. 10 By incorpor- ating divert valves into this system, aliquots of plasma could be directly analyzed without sample preparation. The four-channel LC/MS/MS has re- duced method validation time, increased sample throughput by 4-fold, and afforded adequate sensitivity, precision, and negligible intersprayer cross- talk. 8,9 In protein analysis, an eight-way MUX coupled with a TOFMS analyzer has proved to be a powerful tool to monitor the protein purifica- tion process by screening fractions from preparative ion-exchange chroma- tography with a throughput of 50 protein-containing fractions in less than an hour. 11 A high-pressure gradient parallel pumping system (JASCO PAR-1500) has been developed to conduct high-throughput parallel liquid chromatog- raphy. 12 It is a 10-pump system where two pumps are used to generate a binary gradient and eight pumps to deliver the mixed solvent to eight LC columns. Comparing this system to a conventional system with two pumps or a binary pump for LC gradient and a simple splitter to divide the gradi- ent to eight LC columns, this system can ensure uniform flow rates through each LC column. This system has been used for peptides and combinatorial sample, 12 protein analysis, 13 and bioanalysis. 9 We have optimized an eight-way MUX coupled to a TOFMS analyzer to carry out eight-channel parallel LC/UV/MS analysis of combinatorial libraries 14 in the past 2 years. This system has not only provided the capacity needed for library analysis, but also enabled simultaneous evalu- ation of experimental parameters to expedite the method development process. In this chapter, we discuss the optimization of this system and present a high-throughput protocol for combinatorial library analysis. We also compare the eight-channel parallel LC/UV/MS system to a conven- tional single channel LC/UV/MS system in terms of performance and operation. 9 D. Morrison, A. E. Davis, and A. P. Watt, Anal. Chem. 74, 1896 (2002). 10 L. Yang, T. D. Mann, D. Little, N. Wu, R. P. Clement, and P. J. Rudewicz, Anal. Chem. 73, 1740 (2001). 11 B. Feng, A. Patel, P. M. Keller, and J. R. Slemmon, Rapid Commun. Mass Spectrom. 15, 821 (2001). 12 D. Tolson, A. Organ, and A. Shah, Rapid Commun. Mass Spectrom. 15, 1244 (2001). 13 B. Feng, M. S. McQueney, T. M. Mezzasalma, and J. R. Slemmon, Anal. Chem. 73, 5691 (2001). 14 J. Zhao, D. Liu, J. Wheatley, L. Fang, and B. Yan, Proceedings of the 49th ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, IL, May, 27–31, 2001. [1] high-throughput LC/UV/MS analysis of libraries 5 [...]... could be acquired to define a peak, which resulted in slightly distorted peak shapes in the TICs On the other hand, peak shapes were much better defined in a single-channel system because more than 10 data points could be easily obtained For combinatorial < /b> library analysis, lower sensitivity is not a problem because the parallel synthesis method always produces enough compound for analysis The limited number... compounds from library 2 (LIB2) have also been analyzed to optimize the sample cone voltage Mass spectra of two compounds (LIB2-1 and LIB2-2) at sample cone voltages of 20, 30, and 40 V are shown in Fig 9 MHþ ions are shown as the predominant ions only at 40 V Fragment ions (m/z ¼ 378.3) could be observed with an RA of 100% and 80% for LIB2-1 and LIB2-2 at 30 V MHþ with 30% RA could be found as a minor... assess product purity based on the assumption of similar absorption coefficients at 214 nm for the desired product and the side-products To develop a method for combinatorial < /b> library analysis, we first analyzed six to eight representative compounds from each library under generic LC/UV/MS conditions These conditions would be used for library analysis unless adjustments had to be made based on the study... Tam, and R B Merrifield, Anal Biochem 117, 147 (1981) W Troll and R K Cannan, J Biol Chem 200, 803 (1953) 26 analytical techniques [2] the color observed (red, green, etc.) are particular to certain substrates and may represent false positives Notes The Kaiser test is generally reliable; however, when used to test sterically hindered amines such as aminoisobutyric acid (Aib), results may be difficult... Tests for Solid Phase Synthesis ´ ´ By Jesus Vazquez, Gregory Qushair, and Fernando Albericio Introduction Solid-phase synthesis (SPS)* is limited by a shortage of simple and rapid techniques for reaction monitoring, specifically for functional group transformations The traditional preparation and subsequent analysis (HPLC, *Abbreviations: AliR, alizarin R; BAL, backbone amide linker; DCM, dichloromethane;... Fernando Albericio Introduction Solid-phase synthesis (SPS)* is limited by a shortage of simple and rapid techniques for reaction monitoring, specifically for functional group transformations The traditional preparation and subsequent analysis (HPLC, *Abbreviations: AliR, alizarin R; BAL, backbone amide linker; DCM, dichloromethane; DIEA, N,N-diisopropylethylamine; DME, N,N’-dimethylformamide; DTNB, 5,51-dithio(2nitrobenzoic... Tertiary alcohol Phenol Thiol Carboxylic acid Aldehydew a Tests Kaiser (ninhydrin),a–c trinitrobenzenesulfonic acid (TNBSA),d NF-31,e chloranil,f–h bromophenol blue,i,j nitrophenylisothiocyanate-O-trityl(NPIT),j,k Malachite green isothiocyanate (MGI),j,l Traut’s reagents,j,m and Ellman’s reagents,j,m TNBS,d NF-31,e chloranil,f–h bromophenol blue,i,j MGIi,j TosCl-PNBP,n (1,3,5)-trichlorotriazine (TCT)... and R B Merrifield, Anal Biochem 117, 147 (1981) c W Troll and R K Cannan, J Biol Chem 200, 803 (1953) d W S Hancock and J E Battersby, Anal Biochem 71, 260 (1976) e A Madder, N Farcy, N G C Hosten, H De Muynck, P J De Clercq, J Barry, and A P Davis, Eur J Org Chem 2787 (1999) f T Christensen, Acta Chem Scand B 33, 763 (1979) g T Vojkovsky, Peptide Res 8, 236 (1995) h The chloranil test can also be used... solutions A and B  The tube is then heated at 100 for 3 min A negative test, indicating the absence of free primary amines, is communicated by a yellow or orangepink solution and naturally colored beads A positive test is indicated by a dark blue or purple solution and beads Variations in the darkness of the solution reflect variations in amine concentration while variations in 3 4 V K Sarin, S B H Kent,... found at 10 V for these compounds Parent ions have been broken apart as the sample cone voltage increases from 10 to 40 V A major fragment (m/z ¼ 316.1) with 70% RA could be detected in addition to MHþ (m/z ¼ 423.2, 100% RA) at 40 V for LIB1-1 (Fig 7A) However, more extensive fragmentation was observed for LIB1-2 (Fig 7B) Four fragment ions could be encountered along with MHþ (m/z ¼ 386.2, 90% RA) . California, 94080 xii contributors to volume 369 [1] High-Throughput Parallel LC/UV/MS Analysis of Combinatorial Libraries By Liling Fang,Jiang Zhao,andBing Yan Introduction Combinatorial chemistry. LC/UV/MS analysis of libraries 11 Combinatorial Library Analysis In LC/MS analysis of combinatorial libraries, the MS determines the product identity and its purity is determined by other on-line detection. analogability. A particularly attractive feature of combinatorial chemistry is that when desirable properties are identified, they can often be xiii optimized through second-generation libraries