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Rationally Designed Ligands for Use in Affinity Chromatography 101
mixtures acetone : water (v/v)—1:1, 1:3, 0:1, 1:1, 3:1, 1:0, 0:1. Cyanuric chloride
activated gel is not stored but used immediately for R 1 substitution.
4. Nucleophilic substitution of R 1 : Cyanuric chloride activated gel is divided into
n aliquots, where n is the number of different amines used to synthesize the
combinatorial library. A twofold molar excess (relative to the amount of amination
of the gel) of each amine is dissolved in the appropriate solvent (1 ml/g gel).
The n aliquots are suspended in the previous mixture and incubated at 30°C in
a rotary shaker (200 rpm) for 24 h. After this period, each R 1 substituted gel is
thoroughly washed on a sintered funnel with the appropriate buffer for each amine.
The resulting gel is stored in 20% (v/v) ethanol at 0–4°C or used immediately for
R 2 substitution.
5. Nucleophilic substitution of R 2 : The n amines selected are dissolved in 15 ml of
appropriate solvent. Each amine is in 5 molar excess to the amount of amination of
the gel. Each aliquot of R 1 substituted gel is divided into 5 ml fractions, suspended
in the previous mixture and incubated at 85°C for 72 h. At the end of the synthesis,
the gels are washed with appropriate solvent, weighed and stored at 0–4°C in 20%
(v/v) ethanol.
3.2.2. Solution-Phase Synthesis of Lead Ligands
The conditions vary from case to case and need to be optimized accordingly.
Solution-phase synthesized ligands are characterized by 1 H-NMR, 13 C-NMR
and mass spectroscopy and further immobilized on a solid support (see Note 5).
The synthesis of the PpL-mimic lead ligand, ligand 8/7 was done as shown
in Fig. 3.
3.2.2.1. Synthesis of 4-(4,6-Dichloro-[1,3,5]Triazin-2-Ylamino)
Benzamide
Cyanuric chloride (3.68 g, 20 mmol) was dissolved in acetone (90 ml) and ice
water (20 ml) at 0°C. To this, a mixture of 4-aminobenzamide (2.72 g, 20 mmol)
Fig. 3. Basic steps followed on the solution-phase synthesis of the lead ligand
(ligand 8/7). Details of the synthesis are given in Subheading 3.2.