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Comprehensive Solutions for Purification and Analysis of Combinatorial Libraries

Comprehensive Solutions for Purification and Analysis of Combinatorial Libraries Qunjie Wang and Ronald E. Majors Agilent Technologies Inc . 2850 Centerville Road Wilmington, DE 19808. Content : - Overview of library purification tools - Applications of solid scavengers

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Comprehensive Solutions for Purification and Analysis of Combinatorial Libraries

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  1. Comprehensive Solutions for Purification and Analysis of Combinatorial Libraries Qunjie Wang and Ronald E. Majors Agilent Technologies Inc. 2850 Centerville Road Wilmington, DE 19808

  2. Content: - Overview of library purification tools - Applications of solid scavengers - High throughput HPLC for purification and analysis of libraries

  3. Agilent Technologies- a subsidiary ofHewlett-Packard Co. Chemical Analysis Group: - GC, GC-MS, HPLC, LC-MS, UV-Vis Spectrometer, ICP-MS; - Consumables and Accessories (GC/HPLC columns, and other separation products).

  4. Shopping on the web • www.agilent.com/chem - shopping village -- consumables & accessories -- combinatorial chemistry

  5. Overview

  6. Purification Tools • liquid/liquid extraction • column chromatography • solid scavengers/reagents • solid support synthesis

  7. Liquid/liquid extraction • Mechanism: partition between two immiscible solvents, i.e. water/ether. • Advantage: simple, less expensive. • Limitation: mid-selective; solubility may vary significantly for each component of the library. • Best application: removing salts, highly water soluble species.

  8. Chromatography: Flash/HPLC • Mechanism: partition, non-specific adsorption/desorption • Advantage: general, high purity • Limitation: non-specific; time consuming; high cost • Best application: high purity requirement; unsatisfied with other tools.

  9. Solid Scavengers/Reagents • Mechanism: specific separation by chemical bonding, ion-exchange or adsorption • Advantage: specific, high-throughput, simple to use, low/medium cost • Limitation: availability, variable reactivity towards individual reactant • Best application: removing excess reactants and by-products

  10. Solid Support Synthesis • Mechanism: immobilization /washing • Advantage: higher purity, high-throughput • Limitation: chemistry may be quite different from the analogue in solution; linkers; sequential synthesis only. • Best application: libraries of very large numbers

  11. Solid Scavengers

  12. How do scavengers work • by reaction between scavengers and specific functionality of reactants, i.e. S-NCO/R1NHR2(R1R2NR3) • by ion-exchange, S-SO3H/ RNH2 (R1NHCOR2) • by selective adsorption, SiO2/R3NH+Cl- (R1NHCOR2) S-: solid support

  13. How to choose scavengers • By functionality: electrophiles (S-NCO, S-aldehydes) for amines, nucleophiles; nucleophiles (S-NH2) foracid anhydride, carbonyls; ion-exchangers, S-NR3+X-. “selective between products and impurity” • By support materials: gel-type polystyrene; macroporous polystyrene/DVB (CombiZorb); silica

  14. How to use scavengers • Flow-through method: have the mixture pass through a column, a cartridge or wells packed with a scavenger. - ion-exchange type or very fast reactions; silica-based > best performance. • “Regular” method: add scavengers into the reaction mixture and shake or agitate before filtration • Catch-release • Mix-bed

  15. Flow-Through Method Reaction Block Filter Block prepacked with scavenger Vacuum Collection Block

  16. 96-Wells Blocks

  17. Volume Restraints • For Automated Synthesis Using 96 wells Block: • Blocks hold 2 mL volume: Reaction volume should be at most half of the volume of the well, scavenger only around 500 mL • Collection blocks hold 2.0 mL, but can only safely concentrate about 1.2 mL • So: Scavenge with at most 450 mL volume of scavenger in reaction wells or develop Flow-through method

  18. CombiZorb macroporous scavengers • Based on ultra-pure, spherical silica: S-monoamine(NH2), S-triamine(NH, NH2), S-tertiary amine, S-sulfonic acid, S-aldehyde, S-mercaptan, S-diphenylethylphosphine. • Based on low-swelling macroporous polystyrene/DVB: MP-isocyanate, MP-aldehyde, MP-mercaptan, MP-trisamine(NH, NH2), MP-piperidinomethyl, MP-sulfonyl hydrazide(-NHNH2), MP-sulfonyl chloride

  19. Features and advantages (vs. gel-polystyrene based scavengers) • Silica-based: Ultra pure silica - no interference with reactions. Spherical silica - easy to handle, good through-flow. No-swelling, high density - larger amount for available volume; possible incorporation into different format (membrane, column). Porous structure - solvent independent, good mass transfer of reactants. • Low-swelling Macroporous polystyrene/DVB-based: Low swelling (30% vs. 500% for gel)- larger capacity per volume, easy to handle, possible in different format (membrane, column). Porous structure - broad solvent compatibility.

  20. Types of Silica Standard Commercial Silica Agilent Ultrapure Silica

  21. Performance Comparison

  22. Performance comparison (cont’d)

  23. CombiZorb (silica-based) S: Agilent ultra pure silica

  24. Scavenging Test of S-monoamine

  25. Scavenging Test of S-triamine

  26. Scavenging Test of MP-NCO(2.5 equiv.)

  27. Scavenging Test of MP-CHO (3 equiv.)

  28. Example 1 • Rxn run in 2 mL of Ethyl Acetate, THF, or DMF. Added 200 mL of water, stirred 16 h at RT. • The solution is forced with a pipet bulb through a plug of 450 mL of scavenger in a 2.0 mL tube, and the scavenger is then rinsed with 1.0 mL of solvent. • The eluents are concentrated, redissolved in 4.0 mL of solvent and analyzed by HPLC

  29. Aqueous Cosolvent Sequestering % Acid Remaining

  30. Example 2 - Benzylamine, chlorobenzoyl chloride and S-tertiaryamine were mixed with 2 mL CH2Cl2 at RT and shaken for 1 hour. - S-triamine plus 1 mL acetonitrile was added to the mixture and shaken for 1 h, the solid was filtered off and washed with CH2Cl2 (twice, 0.5 mL each). - Benzyl chlorobenzamide was obtained as a pure product upon solvent evaporation.

  31. Example 3 - Benzylamine and phenyl isocyanate was mixed with 1.5 mL dichloromethane and shaken for 1 hour at RT. - MP-isocyanate and 1 mL MeOH weres added to the reaction mixture, shaken for two more hours; the solid was filtered off and washed with 1 mL MeOH. - Phenyl benzyl urethane was obtained as a pure product upon solvent evaporation.

  32. Example 4

  33. Summary • Two types of porous scavengers (ultra pure silica, low-swelling polystyrene) have been developed with a variety of functionalities. • Preliminary studies demonstrate the major advantages of the new scavengers: - higher capacity for available volume; - broad solvent compatibility; - compatible with different application formats.

  34. References For general applications of scavengers

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