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100 Roque and Lowe
Gly71 and Gly72). Six hydrogen bonds and two salt bridges mediate the interaction
between Fab and the second PpL binding interface.
2. Selection of compounds to be included in the solid-phase combinatorial library:
There is a total of 11 different amino acid residues of the PpL domain (including
interfaces 1 and 2) involved in the interaction with the light chains, which are
generally exposed to the solvent, promoting hydrogen bonds or salt bridges or
being largely buried upon complex formation. These amino acid residues—Ala,
Asp, Gln, Glu, Gly, Ile, Leu, Lys, Phe, Thr and Tyr—were used as the basis
for the design of analog compounds. The analogue compounds all possess an
amine-terminal group to react with cyanuric chloride, and their structures are
equivalent to the side chains of the amino acid residues they mimic. Amine 4
(m-xylylenediamine) resembles a lysine side chain by possessing a–CH 2 NH 2
terminal group but with the addition of an aromatic ring. Similarly, compound 8
(4-amino-benzamide) bears a resemblance to glutamine and asparagine residues
by having a terminal amide group.
3.2. Synthesis of Bis-Substituted-Triazine Ligands
3.2.1. Solid-Phase Combinatorial Synthesis of a Ligand Library
1. Epoxy activation of agarose beads: The required amount of Sepharose® CL-6B is
washed with 40 ml of distilled water/g of gel on a sinter funnel. The washed agarose
is transferred to a 1-l conical flask and 1 ml of distilled water/g of gel added. To
this moist gel, 0.8 ml of 1 M NaOH/ml of gel and 1 ml of epichlorohydrin/ml of
gel are added. The slurry is incubated for 10–12 h, at 30°C on a rotary shaker. The
epoxy-activated gel is washed with 40 ml of distilled water/g of gel on a sinter
funnel and used directly for amination. The epoxy content is determined according
to Note 1.
2. Amination of agarose beads: The washed epoxy-activated gel is suspended in 1
ml of distilled water/g of gel in a 1-l conical flask. About 1.5 ml of ammonia/g
of gel is added, and the gel is incubated for 12 h at 30°C in a rotary shaker. The
aminated gel is washed with 40 ml of distilled water/g of gel on a sinter funnel
and stored in 20% (v/v) ethanol at 0–4°C. The extent of amination is determined
as described in Note 2. Aminated beads can also be purchased from Amersham
Biosciences-GE Healthcare.
3. Cyanuric chloride activation: Aminated agarose is suspended in a 1-l conical flask,
in a solution of acetone/water 50% (v/v), using 1 ml of solution/g of gel. This
mixture is maintained at 0°C in an ice bath on a shaker. Recrystallized cyanuric
chloride (5 molar excess to aminated gel) is dissolved in acetone (8.6 ml/g cyanuric
chloride) and divided into 4 aliquots. The aliquots are added to the aminated gel,
with constant shaking at 0°C and the pH maintained neutral by the addition of 1
M NaOH. Each aliquot is added with an interval of about 30 min, and samples of
gel are taken in order to evaluate the presence of free amines (see Note 3). When
the four aliquots are added, the gel is washed, with 1lofeach of the following