Comprehensive Solutions for Purification and Analysis of Combinatorial Libraries Qunjie Wang and Ronald E. Majors Agilent Technologies Inc. 2850 Centerville.

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

- 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/R1NHR2

(R1R2NR3)

• by ion-exchange, S-SO3H/ RNH2 (R1NHCOR2)

• by selective adsorption, SiO2/R3NH+Cl-

(R1NHCOR2)

S-: solid support

How to choose scavengers

• By functionality: electrophiles (S-NCO, S-aldehydes) for amines, nucleophiles;

nucleophiles (S-NH2) for acid anhydride, carbonyls ; ion-exchangers, S-NR3+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 prepackedwith scavenger

VacuumCollection 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(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

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 CommercialSilica

Agilent Ultrapure Silica

Performance Comparison

Swelling(by THF)

Capacity/v(mmol/mL)

(THF)

Capacity /w(mmol/g)

(THF)

Capacity/w(mmol/g)(MeOH)

Capa.(MeOH)_________

Capa. (THF)

MP 25% 1.4 3 1.8 60%TrisaminePS 500% 0.3-0.5 3-4 <0.3 <10%

MP 25% 0.8 2 1.3 60%PiperidinoPS 400% 0.4 3 <0.3 <10%

MP 40% 0.5 1.6 1.1 70%HydrazidePS 400% 0.1-0.2 1-2 - 20%

MP 30% 0.6 1.8 na naSulfonyl.Chloride PS 500% 0.1-0.2 1-2 na na

Si 0 0.8 0.4 - -PhosphinePS 300% 0.2-0.4 1-1.8 - -

Si 0 1.0(THF/EtOH)

0.5(THF/EtOH)

0.5 >95%Mercaptan

PS 400% 0.2-0.3 1-1.5 - -

PS: gel-type polystyrene based products. na: not applicable.

Performance comparison (cont’d)

MP-isocynate Gela MP-aldehyde Gela

Capacity/vin THF

(mmol/mL)

0.4-0.5 0.1-0.2 0.5-0.7 0.1-0.2

Swellingin THF

30% 600% 30% 600%

Capacity in THF(mmol/g)

1-1.3 1-1.5 1.4-1.6 1-1.6

Capacityin methanol(mmol/g)

0.5-0.6 0.2 0.7 0.2

a. commercial 1% 0r 2% cross-linked polystyrene gel based scavengers

CombiZorb (silica-based)

S-triamine S-monoamine S-sulfonic acid S-tertiaryamine

Capacity/vin THF

(mmol/mL)

1.6-2.1 0.8- 1.2 0.5-0.8 0.8- 1.2

Capacity(mmol/g)

1.2-1.6 0.6-0.9 0.4-0.6 0.6-0.9

Capacityin methanol(mmol/g)

1.2-1.6 0.6-1.0 0.4-0.6 0.5-0.7

S: Agilent ultra pure silica

Scavenging Test of S-monoamine Electrophile Combizorb S-

monoamine(equiv.)1)

Solvent Conditions Scavenged(%) 2)

4-chlorobenzoylchloride

4 CH2Cl2 1 h, 20 oC > 99%

2-phenylbutyrylchloride

4 CH2Cl2 1 h, 20 oC >99%

Phenyl chloroformate 4 CH2Cl2 1 h, 20 oC >99%Chloroacetic anhydride 4 CH2Cl2 1 h, 20 oC > 99%Cyclohexyl isocyanate 2 CH2Cl2 1 h, 20 oC >99%

Phenyl isocyanate 2 CH2Cl2 1 h, 20 oC > 99%Benzaldehyde 3 THF/MeOH

(1:2)1 h, 60 oC > 99%

1) Relative to electrophiles without use of additional base2) Determined by GC

Scavenging Test of S-triamine

Electrophile Combizorb S-triamine(equiv.)1)

Solvent Conditions Scavenged(%) 2)

4-chlorobenzoylchloride

4 CH2Cl2 1 h, 20 oC > 99%

2-phenylbutyrylchloride

4 CH2Cl2 1 h, 20 oC >99%

Phenyl chloroformate 4 CH2Cl2 1 h, 20 oC >99%Chloroacetic

anhydride4 CH2Cl2 1 h, 20 oC > 99%

Phenyl isocyanate 2 CH2Cl2 1 h, 20 oC > 99%Benzaldehyde 3 THF/MeOH

(1:2)1 h, 60 oC > 99%

1) Relative to electrophiles without use of additional base2) Determined by GC

Scavenging Test of MP-NCO(2.5 equiv.)

Nucleophile Solvent Temp oC Time (h) Scavenged (%)1)

benzylamine CH2Cl2 20 0.5 >99benzylamine acetonitrile 20 0.5 >99benzylamine i-PrOH 20 0.5 92benzylamine MeOH 20 0.5 91morpholine THF 20 1 >99

1-methyl piperazine THF 20 1 >99tryptamine THF 20 1 94

phenyl hydrazine THF 20 1 >99aniline THF 50 1 75

1) Determined by GC

Scavenging Test of MP-CHO (3 equiv.) Nucleophile Solvent Additive Temp (oC) Time

(h)Scavenged

(%) 1)

phenylhydrazine THF none 50-60 2 95phenylhydrazine Toluene none 50-60 2 >99phenylhydrazine i-PrOH none 50-60 2 93phenylhydrazine MeOH none 50-60 2 > 99

p-toluenesulfonylhydrazide

THF MeOH 50-60 2 > 99

4-methoxyphenylhydrazine

hydrochloride

MeOH none 50-60 2 >99

Benzylamine THF Aceticacid

50-60 2 >99

Tryptamine THF MeOH 20 3 97

Determined by GC

Example 1

O2N

Cl

O

NH2100 Mol

DIEA

NH

O

100 Mol 50 MolNO2

DIEA HCl

50 MolO2N

Cl

O

50 Mol

200 uL of Water

16 h rt

NH

O

NO2

DIEA HCl

50 MolO2N

OH

O

50 Mol50 Mol

50 Mol

• 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 SequesteringC

on

tro

l

Co

ntr

ol

HP

sil

ica

AP

Sil

ica

P-T

ris

P-D

IEA

P-N

MM

0

20

40

60

80

100

120

Aqueous Cosolvent Sequestering

Ethyl Acetate

Methanol

DMF

% Acid Remaining

NH

O

NO2

DIEA HCl

O2N

OH

O

50 uMol each

Example 2

ClC

O

ClNMe2

NMe2

NH2NMe2HCl

(0.4 mmol)

Cl

PhCH2NH2

C(0.6 mmol)

O

+

Cl

+

NH2

(1.2 meq)

ClCO

NH

NH2HCl

(0.6meq)

ClC

O

PhCH2NH

ClC

O

PhCH2NH

Purity > 99 %

Yield = 95%

: S-tertiaryamine, 0.8meq/g; : S-triamine, 1.4 meq/g.

- 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.

Example 3

NCO

(0.3 mmol)

PhCH2NH2

PhNCO(0.2 mmol)

+

NHCNHCH2Ph

NCO

(0.3 mmol)PhNCO

+

O

NHCNHCH2Ph

O

Ph

NHCNHCH2Ph

O

Ph

: MP-isocyanate, 1mmol/g.

Purity > 99 %

Yield = 87 %

- 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

HN

NH2

O O NN

R

SO2NHNH2

RN

N+

+R1.0 eq. 1.5 eq

MeOH MeOH

R = phenyl, 4- methoxyphenyl, m-tolyl;

Yield > 80%Purity > 95%

RT RT1h 2h

Unlike the gel-type polystyrene based scavengers, the macroporous scavengers can be used in the alcohols with good efficiency.

*

O O

Synthesis of Pyrazoles

2 eq

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

[1] R. J. Booth & J. C. Hodges J. Am. Chem. Soc., 1997, 119, 4882.

[2] D. L. Flynn, et al. J. Am. Chem. Soc., 1997, 119, 4874.

[3] D. L. Flynn, et al. Med. Chem. Res. 1998, 8, 219.

[4] A. T. Merritt. Comb. Chem. High Throughput Screening 1998, 1, 57.

[5] R.J. Booth and J.C.Hodges. Acc. Chem. Res.1999. 32, 18.

For general applications of scavengers