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

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

Agilent Technologies - a subsidiary of Hewlett- 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).

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Overview

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

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.

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.

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

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

Solid Scavengers

How do scavengers work by reaction between scavengers and specific functionality of reactants, i.e. S-NCO/R 1 NHR 2 (R 1 R 2 NR 3 ) by ion-exchange, S-SO 3 H/ RNH 2 (R 1 NHCOR 2 ) by selective adsorption, SiO 2 /R 3 NH+Cl - (R 1 NHCOR 2 ) S-: solid support

How to choose scavengers By functionality: electrophiles (S-NCO, S-aldehydes) for amines, nucleophiles; nucleophiles (S-NH 2 ) for acid anhydride, carbonyls ; ion-exchangers, S-NR 3 +X -. selective between products and impurity By support materials: gel-type polystyrene; macroporous polystyrene/DVB (CombiZorb); silica

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

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

96-Wells Blocks

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 L –Collection blocks hold 2.0 mL, but can only safely concentrate about 1.2 mL –So: Scavenge with at most 450 L volume of scavenger in reaction wells or develop Flow-through method

CombiZorb macroporous scavengers Based on ultra-pure, spherical silica: S-monoamine(NH 2 ), S-triamine(NH, NH 2 ), 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, NH 2 ), MP-piperidinomethyl, MP-sulfonyl hydrazide(-NHNH 2 ), MP-sulfonyl chloride

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.

Types of Silica Standard Commercial Silica Agilent Ultrapure Silica

Performance Comparison

Performance comparison (contd)

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

Scavenging Test of S-monoamine

Scavenging Test of S-triamine

Scavenging Test of MP-NCO (2.5 equiv.)

Scavenging Test of MP-CHO (3 equiv.)

Example 1 Rxn run in 2 mL of Ethyl Acetate, THF, or DMF. Added 200 L of water, stirred 16 h at RT. The solution is forced with a pipet bulb through a plug of 450 L 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

Aqueous Cosolvent Sequestering % Acid Remaining

Example 2 - Benzylamine, chlorobenzoyl chloride and S-tertiaryamine were mixed with 2 mL CH 2 Cl 2 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 CH 2 Cl 2 (twice, 0.5 mL each). - Benzyl chlorobenzamide was obtained as a pure product upon solvent evaporation.

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.

Example 4

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.

References For general applications of scavengers