combinatorial chemistry, part b

To analyze everycompound in a library of 2500 compounds at 3.5 min cycle time requires through-146 h using a single channel LC/UV/MS system... Thissystem could analyze more than 3000 com

Combinatorial chemistry has matured from a field where efforts initiallyfocused on peptide-based research to become an indispensable research toolfor 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 withother 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 ofmolecules with a ‘‘diversity’’ goal in mind, other factors came into playdepending 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 aniterative Darwinian process of divergence and selection Particularly in drugdiscovery, where time is a critical factor to success, combinatorial chemistryoffers the means to test more molecule hypotheses in parallel

We will always be limited to a finite number of molecules that we caneconomically synthesize and evaluate Even with all the advances in automa-tion technologies, combinatorial chemistry, and higher-throughput screens thatimprove our ability to rapidly confirm or disprove hypotheses, the synthesisand screening cycle remains the rate-determining process Fortunately, wecontinue 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 thedrug 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 toidentify optimal conditions Chemical feasibility is at the heart of small mol-ecule discovery and chemotype prioritization since it essentially defines whatcan and cannot be analoged (i.e., analogability) Although analogability is notthe only driving factor, quite often it is overlooked For example, when com-mercially-available compounds or complex natural products are screened, theleads generated are often dropped because of the difficulty to rapidly analogthem 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 combinatorialchemistry is that when desirable properties are identified, they can often be

xiii

optimized through second-generation libraries following optimized syntheticprotocols If this process of exploring truly synthetically accessible chemicalspaces could be automated, then it would open up the exciting possibility ofmodeling 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 anddrugability These are important questions where scientists are now starting togenerate a large-enough body of information on high-throughput syntheticchemistry to begin to more globally understand what is cost-effectively pos-sible Within the biopharmaceutical industry, significant investments in newtechnologies have been made in molecular biology, genomics, and proteomics.However, with the exception of combinatorial chemistry, relatively little hasbeen done to advance the fundamental nature of chemistry in drug discoveryfrom a conceptual perspective

Now, after having gone through the molecule-generating period whereresearch institutions have a large historical compound collection and the pro-liferation of combinatorial chemistry services, the trend is now after makingmore targeted-oriented molecular entities also known as ‘‘focused libraries.’’

An important emerging question is: How can one most effectively make thebest 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 coveredpeptide and peptidomimetic based research with just a few examples of smallmolecule libraries In this volume we have compiled cutting-edge research incombinatorial chemistry, including divergent areas such as novel analyticaltechniques, microwave-assisted synthesis, novel linkers, and synthetic ap-proaches in both solid-phase and polymer-assisted synthesis of peptides, smallmolecules, and heterocyclic systems, as well as the application of these tech-nologies to optimize molecular properties of scientific and commercial interest

Guillermo A MoralesBarry A Bunin

DIVISION OF BIOLOGY CALIFORNIA INSTITUTE OF TECHNOLOGY

PASADENA, CALIFORNIA

FOUNDING EDITORS

Sidney P Colowick and Nathan O Kaplan

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., ment of Small Molecule Drug Discovery, One Amgen Center Drive, Thousand Oaks, California, 91320

Depart-James W Christensen (5), Advanced ChemTech Inc., 5609 Fern Valley Road, Louisville, Kentucky, 40228

Andrew P Combs (12), Incyte ration, Wilmington, Delaware, 19880-0500 Scott M Cowell (16), Department of Chemistry, University of Arizona, Tucson, Arizona, 85721

Corpo-Stefan Dahmen (7), Institut fur 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

Orga-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

Chemistry, University of Arizona,

Tuscon, Arizona, 85721

Eric Healy (5), Advanced ChemTech Inc.,

5609 Fern Valley Road, Louisville,

Kentucky, 40228

Timothy F Herpin (4), Rhoˆne-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.,

California, 94304

Richard Houghten (25), Torrey Pines stitute for Molecular Studies, 3550 Gen- eral Atomics Court, Room 2-136, San Diego, California, 92121

In-Victor J Hruby (16), Department of Chemistry, University of Arizona, Tucson, Arizona, 85721

Christopher Hulme (24), Amgen Inc., 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

De-Ian W James (3), Mimotopes Pty Ltd., 11 Duerdin Street, Clayton, Victoria, 3168, Australia

Wyeth Jones (24), Amgen Inc., ment of Small Molecule Drug Discovery, One Amgen Center Drive, 29-1-B, Thou- sand Oaks, California, 91320

Depart-Patrick Jouin (10), CNRS UPR 9023, CCIPE, 141, rue de la Cardonille, Mont- pellier Cedex 05, 34094, France

C Oliver Kappe (11), Institute of try, Karl-Franzens-University Graz, Heinrichstrasse 28, Graz, A-8010, Austria Steven A Kates (19), Surface Logix, Inc.,

Chemis-50 Soldiers Field Place, Brighton, chusetts, 02135

Massa-Viktor Krchnˇa´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 tology and Oncology, Department of In- ternal Medicine, UC Davis Cancer Center, University of California Davis, Sacramento, California, 95817

Hema-Ruiwu Liu (15, 17), Division of

Hematol-ogy and OncolHematol-ogy, 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), Rhoˆne-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 Padeˇra (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 randa Avenue, Palo Alto, California, 94304

Mi-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 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

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 binational Chemistry, Adolor Corpor- ation, 700 Pennsylvania Drive, Exton, Pennsylvania, 19345

Com-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

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

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

California, 94304

Josef Vagner (16), Department of

Chem-istry, University of Arizona, Tuscon,

Ari-zona, 85741

Jesus Vazquez (2), University of 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

Barce-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

[1] High-Throughput Parallel LC/UV/MS Analysis of

of compounds with the need to rapidly analyze those compounds for theiridentity 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 methodsand mass analyzers.1–3LC/MS*is the most popular technique used in com-binatorial library analysis because it combines separation, molecularweight determination, and relative purity evaluation in a single sample in-jection However, the throughput of conventional LC/MS could not meetthe need to analyze every member in a large combinatorial library in atimely 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.

Copyright 2003, Elsevier Inc All rights reserved.

been used to achieve desirable high speed taking advantage of the low cosity of CO2.5However, the serial LC/MS approach by its nature does notmatch the speed of parallel synthesis Parallel LC/MS is the method ofchoice to increase throughput while maintaining the separation efficiency.

vis-An eight-probe Gilson 215/889 autosampler was incorporated into aquadruple mass spectrometer.6This arrangement enabled the injection ofeight samples (a column from a 96-well microtiter plate) simultaneouslyfor flow-injection analysis/MS (FIA-MS) analysis to achieve a throughput

of 8 samples/min A novel multiplexed electrospray interface (MUX)7was developed in 1999 and became commercially available for parallelhigh-throughput LC/UV/MS analysis The eight-way MUX consists ofeight nebulization-assisted electrospray ionization sprayers, a desolvationgas heater probe, and a rotating aperture It can accommodate all eighthigh-performance liquid chromatograph (HPLC) streams at a reduced flowrate of <100 l/min per stream and conduct electrospray ionization for alleight streams simultaneously Ions are continuously formed at the tip ofeach 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 ionsampling cone, while ions from the other seven sprayers are shielded Eachliquid stream is sampled for a preset time with mass spectra acquired in fullmass range into eight simultaneously open data files synchronized with thespray being sampled With a 0.1-s acquisition time per sprayer and 0.05-sintersprayer delay time, the time-of-flight (TOF) mass analyzer can acquire

a discrete data file of electrospray ion current sampled from each streamover the entire HPLC separation with a cycle time of 1.2 s Therefore,this eight-way MUX-LCT was like having eight individual electrosprayionization (ESI)-MS systems working simultaneously

The MUX interface enables the coupling of parallel liquid raphy to a single mass spectrometer This technology has had a greatimpact in high-throughput LC/MS analysis In drug development, a four-way MUX interface was used on a triple quadrupole mass spectrometer

chromatog-to simultaneously validate LC/MS/MS methods for the quantitation ofloratadine and its metabolite in four different biological matrixes8and 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.

diazepam in rat liver microsomes for in vitro metabolic stability.9The channel LC/MS/MS system was also reported for the quantification of adrug in plasma on both the narrow-bore and capillary scales.10By incorpor-ating divert valves into this system, aliquots of plasma could be directlyanalyzed without sample preparation The four-channel LC/MS/MS has re-duced method validation time, increased sample throughput by 4-fold, andafforded adequate sensitivity, precision, and negligible intersprayer cross-talk.8,9 In protein analysis, an eight-way MUX coupled with a TOFMSanalyzer 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

four-an hour.11

A high-pressure gradient parallel pumping system (JASCO PAR-1500)has been developed to conduct high-throughput parallel liquid chromatog-raphy.12It is a 10-pump system where two pumps are used to generate abinary gradient and eight pumps to deliver the mixed solvent to eight LCcolumns Comparing this system to a conventional system with two pumps

or a binary pump for LC gradient and a simple splitter to divide the ent to eight LC columns, this system can ensure uniform flow rates througheach LC column This system has been used for peptides and combinatorialsample,12protein analysis,13and bioanalysis.9

gradi-We have optimized an eight-way MUX coupled to a TOFMS analyzer

to carry out eight-channel parallel LC/UV/MS analysis of combinatoriallibraries14 in the past 2 years This system has not only provided thecapacity needed for library analysis, but also enabled simultaneous evalu-ation of experimental parameters to expedite the method developmentprocess In this chapter, we discuss the optimization of this system andpresent a high-throughput protocol for combinatorial library analysis Wealso compare the eight-channel parallel LC/UV/MS system to a conven-tional single channel LC/UV/MS system in terms of performance andoperation

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.

System Optimization

The high-throughput parallel LC/UV/MS system consists of an sampler with eight injection probes, two pumps for generating binary gra-dient, eight UV detectors, and an eight-way MUX with a TOF massspectrometer This two-pump arrangement keeps the system simple andcost efficient However, it does not provide pressure regulation for each LCchannel To ensure flow consistency across each channel, we paid specialattention to the selection of tubing, joints, and columns Columns are fromthe same manufacturer and the same batch The tubing is the same lengthinitially for each channel and is further adjusted by checking the flow at theend With these precautions, the flow from this two-pump system could besplit evenly among the eight channels In addition, a standard mixture isanalyzed every 24 injections, and the retention times of these standardsare closely monitored to ensure an even flow across the eight channels

auto-Standards and Flow Monitoring

Five commercial compounds are chosen as standards in system mization and quality control (Fig 1) Theophylline (log P 0.05), 5-phenyl-1H-tetrazole (log P 2.41), reserpine (C log P 3.32), Fmoc-Asp(OtBu)-OH(log P 4.43), and dioctyl phthalate (C log P 8.39)15 were selected for our

opti-15 L Tang, W Fitch, M Alexander, and J Dolan, Anal Chem 72, 5211 (2000).

D Fmoc-Asp(OtBu)-OH

O O

CH3

CH3O

O H

3 C

H3C

N H

N

H H

H

H3COOC

H

H OCH3H

C log P = 3.32

Fig 1 The structures of the five commercial compounds (A to E) used to monitor performance.

standard mixture Although only three compounds have experimentallydetermined log P values (see above) their C log P values range from

0.4 to 8.4, which covers most of the elution range for combinatoriallibrary compounds

Without backpressure regulation for each channel, it is necessary tominimize the flow rate fluctuation over time The relative standard devi-ation (RSD%) in retention time variation among the eight channels over

1 month for compounds A and B was less than 2% and for C and D itwas less than 1% The RSD% for all channels over a 1-month period forcompounds A to D was 3.2, 2.4, 1.6, and 1.5%, respectively Therefore, thissystem is well suited for combinatorial library analysis The UV chromato-grams from channel 5 from different days are shown as an example inFig 2A The retention times of the four compounds (compounds A to D)from all eight channels during a 1-month period are shown inFig 2B.The throughput of this eight parallel LC/UV/MS system is 3200 com-pounds per day for a 3.5-min cycle time per injection of eight samplesunder current optimized conditions It could be further increased by in-creasing the gradient slope and flow rate We have also determined thatfive compounds in the standard mixture gave a linear response from 0.01

to 0.4 mg/ml.16

16 L Fang, M Wang, M Pennacchio, and J Pan, J Comb Chem 2, 254 (2000).

0.0 0.5 1.0 1.5 2.0 2.5 3.0

Fig 2 A selection of UV214 chromatograms from channel 5 on different days and the retention times from the eight-channel system on every day for standard compounds (A to D) monitored over a 1-month period.

The T-Joint Position

A zero dead volume T-joint is used after each UV detector to split the

LC eluent to the MS analyzer and the waste to ensure a flow of 100 l/minentering each channel The position of the T-joint affected the separation

in the total ion chromatogram (TIC) When the T-joint was placed close

to the UV cell (320 mm from the UV cell), the distance between theT-joint and the eight-way MUX interface is 780 mm A sample had totravel 12 s to reach the eight-way MUX inlet after the UV detector at aflow rate of 50 l/min Fmoc-Asp(OtBu)-OH had a full width at half max-imum (FWHM) of 0.05 min when detected at the UV detector (Fig 3C),but the peak width was doubled at the position of the MUX inlet(Fig 3D) Such a peak broadening in TIC could have jeopardized productidentification To minimize these effects, the T-joint was moved as close aspossible to the eight-way MUX inlet With this modification the samplereached the MUX inlet 2 s after leaving the UV detector The FWHM inthe UV and TIC chromatograms were both 0.05 min (Fig 3A and B) Thuspeak delay and broadening were all eliminated

LC Conditions

Unlike a single LC/UV/MS system, reducing solvent consumption is portant in this eight-channel LC/UV/MS system A flow rate of 24 ml/min

im-on eight 4.6 50-mm columns was used initially This operation resulted in

a solvent consumption of 34.5 liters/day To maintain the same separation

Fig 3 UV214and TIC chromatograms of compound D obtained from configurations A (A and B) and B (C and D).

efficiency and minimize solvent consumption, columns with a smaller innerdiameter (i.d.), such as 2.1 mm, were evaluated The standard mixture wasanalyzed at flow rates of 6, 8, 10, 12, 14, and 16 ml/min The LC gradientwas 10–100% B in 3.0 min and 100% B for 0.5 min The chromatograms

inFig 4show the separation results obtained from the original 4.6–mm-i.d.column at 24 ml/min (A) and 2.1-mm-i.d column at 8 ml/min (B), 12 ml/min (C), and 16 ml/min (D) Below 8 ml/min, the very lipophilic dioctylphthalate did not elute The separation was good at 12 ml/min and evenbetter at 16 ml/min A flow rate of 12 ml/min with the 2.1-mm-i.d columnwas sufficient to maintain the separation efficiency, and consumed only halfthe solvent

For a particular library, an optimal LC column needs to be selected.This parallel LC/UV/MS system could evaluate eight different columns in

4 min Five C18columns of 2 50 mm packed with 5-m particles made bydifferent manufacturers were evaluated simultaneously based on the separ-ation efficiency of the standard mixture using trifluoroacetic acid (TFA) oracetic acid as modifier Chromatograms at 12 ml/min using 0.05% TFAfrom each column are shown inFig 5 The Aqua column gave poor peakshape for the early-eluting compound A The Aqua and Luna columnsdid not separate compounds B and C (data not shown) using 0.1% aceticacid The remaining three columns separated the five compounds well

Retention time (min)

An Efficient Rerun Protocol

In a high-throughput analysis mode, variable sample concentrationsometimes leads to inadequate or saturated signals and at times blocking

of the injector ports Therefore, reanalysis of selected samples became anecessity However, due to the rigid design of the Gilson 215 Multiprobeliquid handler, MUX-LCT cannot handle reanalysis efficiently Forexample, when an injector is blocked during an overnight queue, theremay be 24 failed samples on two 96-well plates (Fig 6) Since the 8-probeinjector has to inject an entire column of eight samples for each run, it willtake 24 runs or 108 min to complete the analysis

We have developed a new process to improve the efficiency of samplereanalysis This process includes four steps: data review, replating, reanaly-sis, and data alignment We have also generated an Excel template, aGilson’s Unipoint protocol, and an in-house visual basic program toautomate the process

Raw data processed by the OpenLynx program was first reviewed forconsistency Since the full scale of our analog channel is 2.1 106

, sampleswith a UV peak height over that limit saturate the UV detector Besidesthe detector saturation, samples can also overload the HPLC column andgenerate broad peaks (FWHM > 0.1 min) in HPLC chromatogram Bothtypes of signal saturation were identified, and a dilution factor was esti-mated for each sample Low sample concentration was another reasonfor rerun Failed external standards and sample carryover are indications

of an injector blockage Samples with hydrophilic diversity eluted withthe solvent front using the generic method These samples needed to be

Fig 5 UV chromatograms of the standard mixture separated using 2.1-mm-i.d columns

at 12 ml/min: Polaris (A), Zorbax (B), Omnisphere (C), Luna (D), and Aqua (E).

dissolved in water instead of methanol and should be analyzed using a lower gradient All the above samples were entered into an Excel templatewith a plate view There are three output lists generated automatically bythe template: output to liquid handler for plating, output to MassLynx forreanalyzing, and output for realigning the final data.

shal-A Gilson liquid handler was programmed with the Unipoint software todilute and reformat failed samples A volume of 120 l of solvent (metha-nol unless specified otherwise) was added to each failed vial The solutionwas taken up and released by the sampling needle three times to ensure ef-ficient mixing A fraction of the liquid was then transferred to the targetplate The fraction volume was determined on the Excel template by thedilution factor, for example 40 l for 2:1 dilution Solvent was allowed toevaporate at ambient temperature from the new plate, and 200 l of solventwas then added to each well in the plate The new plate, reformatted andcompressed, was analyzed with the MUX-LCT system using the sample listfrom the template Using this new format, the 24 samples in the exampleabove (Fig 6) were reanalyzed in three runs (13.5 min) instead of 24 runs(108 min) This represents an 8-fold improvement in efficiency

We have also created a visual basic program to modify sample locationinformation Since sample location was hard-coded in the data file, samples

on the reformatted plate were of a different location from their original.Once processed by OpenLynx, these samples will cause a ‘‘multiple injec-tion conflict’’ with the samples that were originally in these locations Thevisual basic program used Microsoft scripting runtime objects to locateeach sample on the reformatted plate It opened the header file, searchedfor the sample location, and replaced it with the original location This pro-gram can also append customized information, such as a new identity after

‘‘cherry picking,’’ into the sample file All added operations, such as platingand alignment, were performed offline of the MUX-LCT With this newprocess, sample reanalysis became much more efficient

F G H E

Combinatorial Library Analysis

In LC/MS analysis of combinatorial libraries, the MS determines theproduct identity and its purity is determined by other on-line detection tech-niques such as UV, evaporative light scattering detection (ELSD), andchemiluminescent nitrogen detection (CLND).17–20 UV detection is usedhere to assess product purity based on the assumption of similar absorptioncoefficients at 214 nm for the desired product and the side-products

To develop a method for combinatorial library analysis, we first lyzed six to eight representative compounds from each library under gen-eric LC/UV/MS conditions These conditions would be used for libraryanalysis unless adjustments had to be made based on the study of theserepresentative compounds

ana-Evaluation of Representative Library Compounds

Five to eight representative compounds were evaluated simultaneouslydue to the parallel nature of the system Depending on the structure of alibrary, this analysis was performed using acetic acid or TFA as modifier

We found that the general LC gradient worked well for most of the libraryexcept in a few cases in which very polar compounds eluted early In thesecases, the sample solvent, solvent gradient, or LC column was varied to op-timize the retention time However, we had to adjust ion optics settings formost libraries to ensure that the MHþion was the predominant ion to makeproduct identification simple We found that sample cone voltage was acritical parameter when all other ion optics parameters were kept constant.This was reasonable because the sample cone separates the ionizationchamber with a pressure near atmospheric pressure from the vacuumregion with a pressure of a few Torr Ions could be fragmented due to col-lision with the gas molecules in this region A higher sample cone voltagewould produce more energetic ions to undergo collision-induced dissoci-ation This eight parallel LC/MS system has dramatically accelerated thisprocess because up to eight compounds can be evaluated simultaneouslyunder the same experimental conditions

Six compounds from library 1 (LIB1) have been analyzed eously at sample cone voltages of 10, 20, 30, and 40 V The mass spectra

simultan-of two compounds (LIB1-1 and LIB1-2) are shown in Fig 7 Only MHþ

17 L Fang, M Demee, T Sierra, J Zhao, D Tokushige, and B Yan, Rapid Commun Mass Spectrom 16, 1440 (2002).

18 L Fang, J Pan, and B Yan, Biotechnol Bioeng Comb Chem 71, 162 (2001).

19 D A Yurek, D L Branch, and M Kuo, J Comb Chem 4, 138 (2002).

20 E W Taylor, M G Qian, and G D Dollinger, Anal Chem 70, 3339 (1998).

100 200 300 400 500 600 700 800 900 1000

m/z 0

100

% 0

100

% 0

100

% 0

313.1 314.1

771.4

387.2

772.5 773.5

PFF115-299-45-A3-30V 87 (1.737) Cm (86:88) TOF MS ES+

438

771.4 386.2

265.1

261.2 287.1 387.2

772.5 773.5

PFF115-299-45-A3-20V 84 (1.677) Cm (83:85) TOF MS ES+

464 386.2

265.2

771.4

387.2

772.4 773.5

PFF115-299-45-A3-10V 91 (1.817) Cm (91:93) TOF MS ES+

416 386.2

845.4 424.2

PFF115-299-40-3-10V 127 (2.527) Cm (126:128) TOF MS ES+

165 423.2

845.4 424.2

846.4

20V

10V

30V 40V

[100% relative abundance (RA)] and 2MHþ (dimer, 50% RA) can befound 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 extensivefragmentation was observed for LIB1-2 (Fig 7B) Four fragment ionscould be encountered along with MHþ (m/z ¼ 386.2, 90% RA) and2MHþ(m/z¼ 771.4, 85% RA) at 40 V In terms of sensitivity, the totalion counts for both of the compounds are lowest at 10 V and highest at

30 V for LIB1-1 and at 20 V for LIB1-2 In general, the higher cone voltageproduces the stronger ion intensity However, higher cone voltage alsocauses fragmentation, which in turn leads to uncertainty in product identi-fication As a compromise for six compounds, the sample cone voltage wasset to 20 V The LC/MS chromatogram and mass spectra of all fivecompounds under optimized conditions are shown inFig 8

Six representative compounds from library 2 (LIB2) have also been lyzed 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

ana-inFig 9 MHþions are shown as the predominant ions only at 40 V ment ions (m/z¼ 378.3) could be observed with an RA of 100% and 80%for LIB2-1 and LIB2-2 at 30 V MHþwith 30% RA could be found as aminor ion at 20 V while doubly charged ions with 100% RA were the majorion With a resolution around 5000, TOFMS made it easy to assign chargestates to each ion in the spectrum Three ions with m/z of 234.6, 378.3, and468.3 found from LIB2-1 at 30 V are displayed in the 3 amu window inFig.10A, B, and C, respectively Charge states could be easily assigned based onthe mass difference between C12 and C13 for each ion observed in the massspectrum A mass difference of a half unit indicated that the ion with m/z of234.6 (Fig 10A) has a charge state of 2 while ions of 378.3 and 468.3have a charge state of 1 since a mass difference of one unit was observed

Frag-It is concluded that product from LIB2 could be easily identified by adoubly charged ion using a sample cone voltage of 20 V or identified by

a singly charged ion at 40 V Detection sensitivity is higher for the doublycharged ion at 20 V than that of the singly charged ion at 40 V Aftermethod development, a set of optimized ion optics settings was saved andused for future analysis of the library along with the suitable LC conditions

Library Analysis

Libraries were analyzed in 10 96-well plate batches Each QC plate tained 88 sample compounds The last column of each plate was reservedfor sampling blank and standard controls Standards were analyzed in

con-100 200 300 400 500 600 700 800 900 1000m/z0

100

% 0

100

% 0

855 2 914.2

988 349.1

697.2

1.11e3 362.1

1.60

1.04

TIC 7.41e3 1.39

TIC 6.06e3 1.09

TIC 1.11e4 1.67

1.12 1.50

TIC 7.41e3 1.76

968 378.3

234.7

235.2

468.4

379.3 469.4

1.28e3 234.6

235.2

468.3 235.7

100

% 0

100

% 0

485.3

PFF107-3-D4-a 41 (0.984) Cm(40:42) 1: TOF MS ES+

647 484.4

378.3 242.7

243.2 394.3 485.4

PFF107-3-D4-a 40 (0.960) Cm(40:42) 1: TOF MS ES+

1.17e3 242.6

every 24 injections during analysis to monitor the performance consistency

of all eight channels The analysis queue was constructed from an Excelspreadsheet and imported into the MassLynx software for execution Afteracquisition, the data were processed using MassLynx in batches Processeddata could be reviewed in OpenLynx by selecting a plate and clicking onthe desired well The UV chromatogram and mass spectrum of the desiredproduct in LIB2, plate26, well D1, are shown as an example inFig 9 Wegenerated an Excel report that included filename, expected molecularweight, purity of desired products at 214 nm, and a plate view with purityindicated for all compounds in the 10-plate batch Library LIB2 wascomposed of 60 plates; it was analyzed in positive ion mode and processed

in six batches The purity distribution of library LIB2 is shown in Fig 11with an average of 80.6% for 5280 compounds measured at 214 nm.Figure 11shows the plate view of all 60 plates According to this protocol,

we have completed more than a half million LC/UV/MS analyses in aperiod of 15 months with two eight-channel MUX-LCT systems

Comparison of the Eight-Channel LC/UV/MS (MUX-LCT) Systemwith a Conventional Single-Channel LC/UV/MS System

The significant advantage of the parallel LC/MS system is its put Because eight LC/UV/MS analyses can be conducted simultaneously,the total analysis time is decreased by a factor of eight To analyze everycompound in a library of 2500 compounds at 3.5 min cycle time requires

through-146 h using a single channel LC/UV/MS system However, it requires only

Average purity 80.6%

0 500 1000 1500 2000

18.2 h to complete this task using an eight-channel parallel LC/UV/MSsystem, and this makes it possible to perform LC/UV/MS analysis on everycompound for all of our libraries In addition, this system also speeds

up method development because it simultaneously evaluates up to eightparameters or variables such as the performance of eight different columns

UV and TIC Chromatograms

An important concern in using an eight-way MUX interface is that theacquisition cycle time (the time required to acquire one data point for eachchannel) is longer, and the data acquisition time per channel is shorter,than for a single-spray system Therefore, the sensitivity might be lowerand the peak shape could be distorted In our current system with a time-of-flight mass spectrometer, the minimum time required for each acquisi-tion cycle is 1.2 s with 0.1 s for data acquisition and 0.05 s for intersprayerdelay The chromatographic baseline peak width was between 5 and 6 s inthe UV chromatograms and between 6 and 7 s in the TICs under generalLC/UV/MS conditions A maximum of five MS data points could be ac-quired 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 easilyobtained For combinatorial library analysis, lower sensitivity is not a prob-lem because the parallel synthesis method always produces enough com-pound for analysis The limited number of data points across an LC peakwas usually not a problem because the MS data were used only to identifythe peak of interest In theory, one or two data points (TOF mass spectra)should be sufficient to confirm the expected molecular weight The productpurity was obtained from the UV chromatogram, where the number ofdata points was sufficient to ensure excellent peak shape and precision

Data Acquisition Using Positive and Negative Ionization

In a single-spray system, it is common to analyze samples in both tive and negative ion modes by switching polarity during a single data ac-quisition This practice makes the best use of precious MS time andidentifies products by their presence in both positive and negative ionforms Both positive and negative ESI modes are available for the eight-channel MUX-LCT system However, the polarity change within a singledata acquisition would make the cycle time much longer Therefore, weprefer to analyze samples using a single polarity, and conduct a separateexperiment with the other polarity if necessary With this arrangement,high-throughput LC/MS analysis with both positive and negative mode

posi-is available

Sample Rerun

For a conventional single-channel LC/UV/MS system, a single satisfied well could be easily reanalyzed In the eight parallel LC/UV/MSsystem, the rerun procedure was different from that of the single-spraysystem If problems were found in a single channel, such as retention timeshift or channel blockage, 12 wells in a row would fail and the whole platehad to be reanalyzed We have developed a rerun protocol that made theparallel LC/MS analysis as efficient as the single-channel system

un-Operation and Maintenance

In the eight-channel parallel LC/UV/MS system, a standard mixturewas analyzed every 24 injections This was indispensable for the operation.The variation of the retention time across eight channels was monitoredclosely to ensure consistency for the eight channels A significant retentiontime shift indicated problems that usually could be overcome by replacingthe frit in the precolumn filter A diminished peak area or a change in peakshape of standards indicated column deterioration We started with eightcolumns from the same batch for sample analysis Deteriorated columnswere replaced individually This practice gave us satisfactory analysis datafor combinatorial library analysis with minimal cost

We anticipated difficulty in maintaining and troubleshooting an channel parallel system because the problems in the autosampler, LCcolumns, UV detectors, and MS interface would be multiplied by eight

eight-In fact, with the convenience of simultaneous analysis of the other sevenchannels, the diagnosis and troubleshooting were made easier The com-plete system was easily divided into four functions: injection, separation,

UV detection, and MS detection By running the standard mixture on eightchannels then switching channels at different function sites and rerunningthe standard mixture, problems were easily isolated Fixing the problemswas exactly the same as for the single-spray system

Conclusion

We have optimized an eight-channel parallel LC/UV/MS (MUX-LCT)system for high-throughput LC/UV/MS analysis of large combinatorial lib-raries Since the LC gradient is divided into eight LC columns by a simplesplitter, the flow fluctuation has been continuously monitored and minim-ized using a standard mixture during analysis to ensure performance con-sistency among the eight channels To preserve the separation integrity inthe total ion chromatogram, the zero dead volume T-joint used to split theflow (after UV detection) should be best placed as close to the eight-way

MUX inlet as possible A flow rate of 12 ml/min on eight 2.1 50 mm laris C18 columns was optimal for general purposes in our study Thissystem could analyze more than 3000 compounds per day for a gradientseparation with a cycle time of 3.5 min.

Po-We have carried out more than half a million LC/UV/MS analyses in 15months using two eight-channel parallel LC/UV/MS systems We foundthat it was beneficial to evaluate a few representative compounds fromeach library and optimize ion optics to make product identification simpleand reliable This parallel system has enabled simultaneous evaluation ofeight compounds and significantly improved the speed of optimization.The identity and purity of every single product could be obtained fromOpenLynx in 10 96-well plate per batch process and transferred into anExcel spreadsheet for the entire library Compared with a single-channelLC/UV/MS system, the parallel LC/UV/MS system has the advantages ofhigh throughput and simultaneous evaluation of eight parameters

*Abbreviations: AliR, alizarin R; BAL, backbone amide linker; DCM, dichloromethane; DIEA, N,N-diisopropylethylamine; DME, N,N’-dimethylformamide; DTNB, 5,51-dithio(2- nitrobenzoic acid) or Ellman’s reagent; Et 3 N, triethylamine; EtOH, ethanol; HOAc, acetic acid; EtOAc, ethyl acetate; Hex, mixture of hexane isomers plus methylcyclopentane; HPLC, high-pressure liquid chromatography; MeOH, methanol; MG, malachite green; MS, mass spectrometry; NMM, N-methylmorpholine; NMP, N-methylpyrrolidinone; Purpald, 4-amino-3- hydrazino-5-mercapto-1,2,4-triazole; SPS, solid-phase synthesis; TCT, trichlorotriazine; THF, tetrahydrofuran; TLC, thin-layer chromatography; TNBSA, trinitrobenzenesulfonic acid; TosCl-PNBP, p-tosylchloride p-nitrobenzylpyridine; TRIS, tris(hydroxymethyl) aminomethane.

Copyright 2003, Elsevier Inc All rights reserved.

MUX inlet as possible A flow rate of 12 ml/min on eight 2.1
50 mm laris C18 columns was optimal for general purposes in our study Thissystem could analyze more than 3000 compounds per day for a gradientseparation with a cycle time of 3.5 min.

Po-We have carried out more than half a million LC/UV/MS analyses in 15months using two eight-channel parallel LC/UV/MS systems We foundthat it was beneficial to evaluate a few representative compounds fromeach library and optimize ion optics to make product identification simpleand reliable This parallel system has enabled simultaneous evaluation ofeight compounds and significantly improved the speed of optimization.The identity and purity of every single product could be obtained fromOpenLynx in 10 96-well plate per batch process and transferred into anExcel spreadsheet for the entire library Compared with a single-channelLC/UV/MS system, the parallel LC/UV/MS system has the advantages ofhigh throughput and simultaneous evaluation of eight parameters

*Abbreviations: AliR, alizarin R; BAL, backbone amide linker; DCM, dichloromethane; DIEA, N,N-diisopropylethylamine; DME, N,N’-dimethylformamide; DTNB, 5,51-dithio(2- nitrobenzoic acid) or Ellman’s reagent; Et 3 N, triethylamine; EtOH, ethanol; HOAc, acetic acid; EtOAc, ethyl acetate; Hex, mixture of hexane isomers plus methylcyclopentane; HPLC, high-pressure liquid chromatography; MeOH, methanol; MG, malachite green; MS, mass spectrometry; NMM, N-methylmorpholine; NMP, N-methylpyrrolidinone; Purpald, 4-amino-3- hydrazino-5-mercapto-1,2,4-triazole; SPS, solid-phase synthesis; TCT, trichlorotriazine; THF, tetrahydrofuran; TLC, thin-layer chromatography; TNBSA, trinitrobenzenesulfonic acid; TosCl-PNBP, p-tosylchloride p-nitrobenzylpyridine; TRIS, tris(hydroxymethyl) aminomethane.

Copyright 2003, Elsevier Inc All rights reserved.

MS, etc.) of resin cleavage products are time-consuming processes, hencealternative methods are desirable The use of colorimetric functional grouptests, wherein aliquots of resin are mixed with stock solutions and changes

in solution/resin color are used to indicate the presence or absence of tional groups on the resin, was initiated by Kaiser et al.1in the use of nin-hydrin to test for primary amines in the SPS of peptides Today organicchemists have at their disposal an ever-broadening array of tests, bothqualitative and quantitative, for alcohols, aldehydes, amines, carboxylicacids, and thiols We present a literature overview of the most widely usedqualitative tests including instructions on reagent preparation and storage,experimental protocol, and the scope and limitations of each test.2 Themajority of these tests can be performed in less than 10 min with simplelaboratory equipment and minimal reagent preparation We also reportthe results of experiments to determine the functional group response ofeach test using amino acids as representative organic compounds

func-It was our intention to provide a central reference for the most commonqualitative tests with a special emphasis on substrate compatibility, namelyfunctional group interference For example, it is known that certain aminoacids may give unusual results for a given test (such as cysteine with theninhydrin test) and some of the original colorimetric test publications in-clude brief reports on the potential for functional group interference (falsepositives) for a given test To determine the utility of each test in the pres-ence of multiple functional groups, each of the summarized colorimetrictests was applied against a broad range of amino acids The aim of this ex-ercise was to determine the universality of each test, and to identify andreport those cases with unusual results The amino acids were tested forthe presence of each functional group under all possible levels of amineand lateral chain protection, thus enabling us to determine the extent towhich chemical interference by other functional groups could affect eachtest (see Scheme 1) Table I summarizes qualitative colorimetric testsreported in the literature for various organic functional groups

We also investigated the use of each test at medium (approximately0.5 mmol/g) and low (approximately 0.025 mmol/g) resin loading

General Experimental Procedures

Resin (polystyrene-based resin, 1% divinylbenzene, 100–200 mesh)substitution and amino acid deprotection were carried out in disposable

1 E Kaiser, R L Colescott, C D Bossinger, and P Cook, Anal Biochem 34, 595 (1970).

2 To the best of our knowledge, only one other review covering some of the methods described herein exists in the literature: C Kay, O E Lorthioir, N J Parr, M Congreve,

S C McKeown, J J Scicinski, and S V Ley, Biotechnol Bioeng 71, 110 (2001).

syringes fitted with polypropylene filter discs using standard solid-phasepeptide synthesis procedures For the majority of the tests described, theexperimental protocols were adapted with minor changes from the originalpublications, none of which we feel jeopardizes the essence of each test(i.e., the underlying chemistry) In the majority of cases, tests were per-formed immediately after preparation of each resin by aliquoting the resininto equivalent portions using the following technique: to the syringe con-taining the master quantity of resin is added dichloromethane (DCM, ca.

1 ml/100 mg resin), the resin is agitated with a pipetter by continuouslytaking up and ejecting a small volume in order to create a uniform suspen-sion, and the desired volume of suspension is quickly removed and trans-ferred to an Eppendorf tube or glass vial and allowed to air dry Thistechnique is more effective than dispensing dry resin with a spatula since

it is faster and more precise for tiny aliquots (1–5 mg) of resin Eppendorftubes (2 ml) were used for all tests except the TosCl-PNBS, Kaiser,Va´zquez, and Purpald tests Disposable glass tubes (800 l) were usedfor the Kaiser and Va´zquez tests, a disposable syringe (1 ml) fitted with apolypropylene filter disc was used for the TCT, Methyl red, and Purpald

Scheme 1 Amino acids were tested at all possible levels of protection This enabled us to differentiate between test results caused by a given free functional group and results that may have been caused by other chemical moieties in the molecule.

TABLE I Summary of Qualitative Colorimetric Test

Primary aliphatic

amine

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,mSecondary aliphatic amine TNBS,dNF-31,echloranil,f–hbromophenol blue,i,jMGIi,jPrimary alcohol TosCl-PNBP,n(1,3,5)-trichlorotriazine (TCT) with fluorescein,

Alazarin R, or fuchsin,o,pSecondary alcohol TosCl-PNBP,nTCT-fluorescein, Alizarin R, or fuchsino,pTertiary alcohol Diphenyldichlorosilane-methyl redq

Phenol TosCl-PNBP,n,rTCT-fluorescein, Alizarin R, or fuchsin,o,p

diphenyldichlorosilane-methyl redqThiol Ellman’s reagents,t

Carboxylic acid Malachite green, u Cystamine-Ellman’s reagent j,v

Aldehyde w Va´zquez ( p-anisaldehyde), x Purpald y

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 to selectively react with primary amines (see experimental section).

i V Krchna´k, J Va´gner, P Safar, and M Lebl, Collect Czech Chem Commun 53, 2542 (1988).

j This test was not reviewed for this publication.

k S S Chu and S H Reich, Bioorg Med Chem Lett 5, 1053 (1995).

l A Shah, S S Rahman, V de Biasi, and P Camillero, Anal Commun 34, 325 (1997).

m T T Ngo, Appl Biochem Biotechnol 13, 213 (1986).

J P Baydal, A M Cameron, N R Cameron, D M Coe, R Cox, B G Davis, L J Oates,

G Oye, and P G Steel, Tetrahedron Lett 42, 8531 (2001).

u M E Attardi, G Porcu, and M Taddei, Tetrahedron Lett 41, 7391 (2000).

v T T Ngo, Appl Biochem Biotechnol 13, 207 (1986).

w An aldehyde (BAL) linker was used as a model for this functional group.

x J Va´zquez and F Albericio, Tetrahedron Lett 42, 6691 (2001).

y J J Cournoyer, T Kshirsagar, P P Fantauzzi, G M Figliozzi, T Makdessian, and

B J Yan, J Comb Chem 4, 120 (2002).

tests, and the TosCl-PNBP test was performed on TLC plates (silica gel,aluminum backed) NF-31 test sample tubes were heated directly in apreheated, multiwell aluminum block Kaiser and Va´zquez test sampletubes were heated in a preheated sand bath inside of a laboratory oven.Heating of the silica plates for the TosCl-PNBP test was performed using

a laboratory heat gun on high setting

Aliphatic Amines

Test: Kaiser (Ninhydrin)1,3,4

Application: detection of primary amines

Test time: 4 min

Reagent preparation time: 1 day

Recommended storage time: 1 month at room temperature inlight-proof containers (such as amber bottles)

Required Reagents

Ninhydrin dissolved in ethanol

Phenol dissolved in ethanol

aq KCN dissolved in pyridine

Preparation of Reagent Solutions

Reagent Solution A Phenol (40 g) in added to EtOH (10 ml) and themixture is heated until complete dissolution of the phenol A solution ofKCN (65 mg) in water (100 ml) is added to pyridine (freshly distilled overninhydrin, 100 ml) Both solutions are stirred for 45 min with AmberliteMB-3 (Merck), filtered, and mixed

Reagent Solution B A solution of ninhydrin (2.5 g) in absolute EtOH(50 ml) is prepared and maintained in a light-proof container, preferablyunder inert atmosphere

Experimental Procedure The resin is washed with appropriate solventsand a small portion (ca 1–5 mg) is transferred to a small glass tube Tothis tube are added three drops of each of the reagent solutions A and B.The tube is then heated at 100 for 3 min A negative test, indicating theabsence of free primary amines, is communicated by a yellow or orange-pink solution and naturally colored beads A positive test is indicated by

a dark blue or purple solution and beads Variations in the darkness ofthe solution reflect variations in amine concentration while variations in

3 V K Sarin, S B H Kent, J P Tam, and R B Merrifield, Anal Biochem 117, 147 (1981).

4 W Troll and R K Cannan, J Biol Chem 200, 803 (1953).

the color observed (red, green, etc.) are particular to certain substrates andmay represent false positives.

Notes The Kaiser test is generally reliable; however, when used to teststerically hindered amines such as aminoisobutyric acid (Aib), results may

in the case of secondary amines or sterically hindered amines such as Aib

We found that at lower levels of resin functionalization a clear positivewas difficult to observe

Test: TNBSA (trinitrobenzenesulfonic acid)5

Application: detection of primary/secondary amines

Test time: 5 min

Reagent preparation time: minutes

Recommended storage time: up to 1 month refrigerated storageRequired Reagents

A 1% (w/v) solution of TNBSA in DMF

A 10% solution of N,N-diisopropylethylamine (DIEA) in DMFExperimental Procedure The resin is washed with MeOH and a smallportion (1–3 mg) is transferred to an Eppendorf tube and suspended inDMF To this tube is added 1 drop of each of the above solutions The solu-tion is left for 5 min at room temperature The resin is washed extensivelywith DMF The presence of free amines is indicated by orange or red beads.Notes The TNBSA test was found to be efficient for primary amines,including sterically hindered amines as seen in our probes with the tertiaryamine of Aib

We also found that at lower levels of resin functionalization a clearpositive was difficult to observe

Test: NF-316

Application: detection of primary/secondary amines

Test time: 10 min

Reagent preparation time: 1 week preparation

Recommended storage time: up to 1 month at 4

5 W S Hancock and J E Battersby, Anal Biochem 71, 260 (1976).

6 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).

Saponification The purified product (1g, 1.0 eq.) from step 1 andKOH (870 mg, 5 eq.) are dissolved in MeOH–toluene [(5:1), 60 ml] Thesolution is stirred and brought to reflux (ca 85) for 90 min The reaction

is allowed to cool and a red precipitate is observed The cooled reactionmixture is concentrated by rotary evaporation to a volume of 10 ml Then10% aq HCl (10 ml) is added, followed by water (25 ml) The precipitate

is extracted with DCM and the organic phase is washed with water anddried on anhydrous MgSO4 The mixture is filtered, and the filtrate isconcentrated by rotary evaporation and the product purified by columnchromatography

Condensation with p-Nitrophenol The product (500 mg, 1.0 eq.)from step 2 and p-nitrophenol (178 mg, 1.0 eq.) are dissolved in DCM(26 ml) and pyridine (22 ml) is then added The aforementioned solution

is maintained at 15, a solution of POCl3 (222 l, 1.8 eq.) in DCM(2 ml) is added dropwise, and the solution is then left to react overnight

at room temperature The crude reaction mixture is then washed with aq.saturated NaHCO3and brine and dried over anhydrous MgSO4 The crudeproduct is concentrated by rotary evaporation and purified by columnchromatography [silica gel, EtOAc-Hex (1:9)]

Experimental Procedure The resin is washed with methanol and a smallportion (1–3 mg) is transferred to an Eppendorf tube To this tube is addedNF-31 solution (0.002 M in acetonitrile, 200 l) The tube is heated in analuminium dry heating block at 70 for 8 min The resin is washed exten-sively with MeOH (3
), DMF (3
), and DCM (3
) The presence of free

amines is indicated by red-colored beads whereas a negative test yieldsnaturally colored beads.

Notes The NF-31 test was found to be highly sensitivite for primary andsecondary amines

During our probes of this test we found that false positives are given ifthe resin is not washed thoroughly with the appropriate solvents

Test: Chloranil7,8

Application: detection of primary/secondary amines

Test time: 5 min

Reagent preparation time: minutes

Recommended storage time: up to 1 month refrigerated storageRequired Reagents

Acetaldehyde (for detection of primary or secondary amines) oracetone (for detection of secondary amines)

Saturated solution of chloranil in toluene

Experimental Procedure The resin is washed with MeOH and a smallportion (1–3 mg) is transferred to a small glass tube To this tube is addedacetaldehyde (primary or secondary amines) or acetone (secondaryamines) (200 l) followed by the chloranil solution (50 l) The solution

is shaken at room temperature for 5 min The presence of free amines is dicated by a green- or blue-colored solution Negative samples register asyellow, amber, or brown

in-Notes The presence of a secondary amine should be confirmed by apositive result obtained for the secondary test and a simultaneouslyobtained negative result for the primary test Likewise the Kaiser test can

be used in place of the primary amine version of the chloranil

This test gave excellent results in both of its forms (testing for primaryand for secondary amines); a clear positive was observed even at low levels

of resin functionalization for the sterically hindered Aib

Alcohols

Test: TosCl-PNBP9

Application: detection of alcohols and phenols

Test time: ca 5 min

Reagent preparation time: 5 min

Recommended storage time: no more than 2 weeks at 4

7 T Christensen, Acta Chem Scand B 33, 763 (1979).

8 T Vojkovsky, Peptide Res 8, 236 (1995).

9 O Kuisle, M Lolo, E Quin˜oa´, and R Riguera, Tetrahedron 55, 14807 (1999).

Required Reagents

A solution of p-toluenesulfonyl chloride (0.12 M) in toluene(solution 1)

A solution of p-nitrobenzylpyridine (0.30 M) in toluene (solution 2)

A 10% (v/v) solution of piperidine in CHCl3(solution 3)

Experimental Procedure The resin is washed with DCM A small tion (3–5 mg) of resin is deposited onto a silica plate by pipette as a DCMsuspension The suspension should be pipetted quickly so that it forms adisperse disc (not a mound) Once dry, the resin is treated with one drop

por-of solution 1 and one drop por-of solution 2 The plate is then heated with aheat gun by swaying the plate in front of the gun from a distance of ap-proximately 5 cm for approximately 1 min A yellow color should appearand then disappear within the heating time, leaving the resin similar to orslightly darker than its natural color At this point a drop of solution 3 isadded to the resin sample on the plate Purple coloration of the beadsindicates the presence of free hydroxyl groups (light pink or purple atlow concentration, dark purple at high concentration)

Notes To get reliable results, concentrations of reagents should beapproximately four times higher than that reported in the original paper

To perform several tests on one silica plate, the resin spots should bedeposited approximately 1–2 cm from each other in each direction

It is always advisable to carry out control tests, both a positive (a resinbearing either a free alcohol or phenol) and a negative (ideally, an acety-lated hydroxy resin) In this case, heating of the plate should be carriedout until the positive resin control takes an orange-red color

We found this test to be highly dependent on the quality of the solutionsused Solutions stored at room temperature for prolonged periods of timegave almost 100% false positives

Although there are conflicting reports in the literature on the utility ofthis test for phenols,9,10in our hands the tests for the phenol of Tyr gave theexpected positive results

Test: TCT-(Fluoresceine, Alizarin R, or Fuchsin)11,12

Application: Detection of alcohols

Test time: ca 30 min

Reagent preparation time: minutes

Recommended storage time: No more than 2 weeks at 4

10 B A Burkett, R C D Brown, and M M Meloni, Tetrahedron Lett 42, 5773 (2001).

11 M E Attardi, A Falchi, and M Taddei, Tetrahedron Lett 41, 7395 (2000).

12 M E Attardi, A Falchi, and M Taddei, Tetrahedron Lett 42, 2927 (2001).

1,3,5-Trichlorotriazine (TCT)

N-Methylmorpholine (NMM)

Fluoresceine, Alizarin R, or Fuchsin

Experimental Procedure The procedure is composed of six steps:

1 A few milligrams of resin is placed in a small glass tube and washedwith DMF

2 DMF (3 ml) is added followed by NMM (1 ml) and TCT (5 mg)

3 The tube is heated at 70 for 20 min

4 The solution is pipetted off and the resin is washed thoroughly withDMF

5 DMF (3 ml) is then added to the resin followed by AliR (5 mg) (or

3 ml of a 0.025% solution of fuchsin or fluoresceine in NMP) andNMP (1 ml)

6 After 5 min the resin is washed thoroughly with DMF until a clearsolution is obtained, then washed with THF and finally with DCM

A positive test is communicated by red beads for the AliR test andgreen or fluorescent beads in the case of the fluoresceine test

Notes A drawback found in the use of this test is the formation of awhite precipitate during the activation of alcohol with the triazine

If fluoresceine is used, the resin beads must be viewed with ultravioletlight as visible light is not sufficient to determine results

Ambiguous results were often obtained with this test, above all withphenols such as the one of tyrosine

This test also yields positive results in the presence of other philes such as primary and secondary amines, carboxylic acids, and thiols.Test: Diphenyldichlorosilane-Methyl red10

nucleo-Application: Detection of alcohols

Test time: ca 25 min

Reagent preparation time: minutes

Recommended storage time: Up to 1 month at room temperatureRequired Reagents

10% triethylamine (Et3N) in dry DCM

Diphenyldichlorosilane

0.75% (w/v) of methyl red in DMF

Experimental Procedure A few milligrams of resin are moistened with

a solution of 10% Et3N in anhydrous DCM (200 l) and treated with phenyldichlorosilane (100 l) for 10 min The resin is then filtered, washed

di-twice with 10% Et3N in anhydrous DCM, at which point a 0.75% (w/w) lution of methyl red in DMF (300 l) is added and the resin allowed toshake for 10 min The resin is filtered, washed with DMF (5
1 min),and then with DCM (5
1 min).

so-A positive test is indicated by orange beads, which become more dish with time In the case of ambiguous results, the beads can be treatedwith formic acid and will take on a purple color in the case of a positiveresult

red-Notes This test proved to be satisfactory not only for phenols but forprimary and secondary alcohols as well

In some cases, light purple beads may be observed even without theaddition of formic acid as a true positive

We strongly recommend the use of a blank control run in parallel withthe sample to be tested as the colorant used in this test readily stays trappedwithin resins

This test also yields positive results in the presence of othernucleophiles such as primary and secondary amines, carboxylic acids, andthiols

Thiols

Test: Ellman’s reagent13–15

Application: Detection of thiols

Test time: 4 min

Reagent preaparation time: 5 min

Recommended storage time: up to 1 month at 4

Required Reagent

5,50-Dithio(2-nitrobenzoic acid), ‘‘DTNB,’’ or ‘‘Ellman’s reagent’’dissolved in aq TRIS solution (1 M, pH 8)

Experimental Procedure To a suspension of an aliquot of resin in DMF

is added three or four drops of the DTNB solution The solution is shaken

at room temperature for 3 min The presence of free thiols is indicated by ayellow-orange color

Notes This test, an adaptation of the original Ellman’s reagent test,worked well for the detection of free thiol groups

13 G L Ellman, Arch Biochem Biophys 82, 70 (1959).

14 J P Baydal, A M Cameron, N R Cameron, D M Coe, R Cox, B G Davis, L J Oates,

G Oye, and P G Steel, Tetrahedron Lett 42, 8531 (2001).

15 M Royo, unpublished results (1991).

Carboxylic Acids

Test: Malachite green (MG)16

Application: Detection of carboxylic acids

Test time: ca 5 min

Reagent preparation time: minutes

Recommended storage time: up to 1 month at 4

Required Reagents

A 0.25% (w/v) solution of malachite green dissolved in ethanol Et3NExperimental Procedure A small portion (1–3 mg) of resin is trans-ferred to an Eppendorf tube and washed with MeOH To this tube is addedthe malachite green solution (1 ml) followed by two drops of Et3N The so-lution is left to stand at room temperature for 3 min and the resin is washedextensively with MeOH The presence of free carboxylic acids is indicated

by green beads

Notes We found this test to be highly reproducible, but the test time iscrucial to obtaining accurate results We observed false positives whenresin samples were left in the malachite green solution for more than

15 min, and a loss of color when green beads are left in MeOH for morethan 15 min

Aldehydes

Test: Va´zquez17

Application: Detection of aldehydes

Test time: 5 min

Reagent preparation time: ca 5 min

Recommended storage time: up to 1 month at room temperatureRequired Reagents

A solution of EtOH (88 ml), H2SO4(9 ml), and HOAc (1 ml)p-Anisaldehyde

Experimental Procedure A solution of p-anisaldehyde (26 l) in thefirst reagent (1 ml) is made A small portion (1–3 mg) of resin is transferred

to a small glass tube and washed with MeOH To this tube is added the vious EtOH/H2SO4/HOAc solution (500 l) The solution is heated in asand bath at 110 for 4 min The presence of free aldehydes is indicated

pre-by orange- to burgundy-colored beads

16 M E Attardi, G Porcu, and M Taddei, Tetrahedron Lett 41, 7391 (2000).

17 J Va´zquez and F Albericio, Tetrahedron Lett 42, 6691 (2001).

Notes This test is very reliable This test is compatible with acid-labileresins such as the Wang and chlorotrityl resins While in the case of theWang resin similar results were obtained, in the case of the chlorotritylresin solutions also became colored, indicating cleavage of the aldehyde(BAL handle) from the resin.

Test: Purpald18

Application: Detection of aldehydes

Test time: 25 min

Reagent preparation time: minutes

Recommended storage time: N/A (see Notes)

or purple beads At lower values of resin loading, a longer air oxidationtime may be required for color to develop (up to 20 min)

Notes Due to the instability of Purpald in solution, it is imperative thatonly freshly prepared reagent solution be used

Conclusions and Summary

Results obtained in the application of these tests are summarized inTable II We have encountered some variations in the reproducibility andaccuracy of some tests Due to the numerous factors that can influence col-orimetric test results (e.g., test reagent stability, resin type, functionalgroup interference, and lability of protecting group) we highly recommendperforming a positive and a negative control for any test applied to a newsynthesis We also emphasize the importance of reagent solution purity onthe outcome of test results, hence we strongly encourage the use of cor-rectly prepared and carefully stored reactants To minimize false results

18 J J Cournoyer, T Kshirsagar, P P Fantauzzi, G M Figliozzi, T Makdessian, and B J Yan, J Comb Chem 4, 120 (2002).

TABLE II Summary of Results Obtained in the Application of the Testsa

Colorimetric test

Functional group Kaiser TNBS NF-31

Chloranil first amine

Chloranil second amine

PNBP TCT

TosCl-Methyl red Ellman MG Va´zquez Purpald Primary alcohol / / / / / þþ/þ þþ/þ þþ/þ / / / /

a A/B, A is the result obtained for a 100% loading resin and B is the result for a 5% loading resin; þþ, intense; þ, less intense; þ, not clear;

, no difference with a blank control.