Comprehensive Solutions for Purification and Analysis of Combinatorial Libraries Qunjie Wang and Ronald E. Majors Agilent Technologies Inc. 2850 Centerville.
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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).
Shopping on the web
• www.agilent.com/chem
- shopping village -- consumables & accessories
-- combinatorial chemistry
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
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