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96 Roque and Lowe
and RasMol V2.7.1.1 (http://www.umass.edu/microbio/rasmol/). Protein X-ray and
nuclear magnetic resonance (NMR) crystallographic structures are available from
the Brookhaven database (http://www.rcsb.org/pdb/), which possesses over 33,000
entries. For the development of a PpL mimic, we have utilized the crystal structure
of the complex between a single PpL domain and a human antibody Fab fragment
(Fab 2A2; human V L -1) refined to 2.7 Å (PDB code: 1HEZ) (10).
2.2. Synthesis of Bis-Substituted-Triazine Ligands
1. Sepharose® CL-6B: Product available from Amersham Biosciences-GE
Healthcare (Piscataway, NJ), which can be obtained as a suspension of beads in
a 20% (v/v) aqueous ethanol solution. Must be stored at 4°C, avoiding periods
of dryness. Agitation of gel suspensions, when required, should be made with an
orbital shaker and not using a magnetic stirrer.
2. Epichlorohydrin (1-chloro-2,3-epoxypropane): Widely available chemical (a high
purity (+99%) or equivalent should be used) which is utilized to epoxy-activate
the Sepharose® CL-6B beads or other surfaces. It is a very unstable compound
and must be stored in an anhydrous environment at 0–4°C. The extent of epoxy
activation of beads can be determined (see Note 1). Hazards: Flammable, poison,
toxic by inhalation, and in contact with skin and if swallowed may cause cancer.
Toxicity data: LD50 90 mg/kg oral, rat. Note: Should be handled in a fume hood
with safety glasses and gloves and treated as a possible cancer hazard.
3. Ammonia aqueous solution (35% (v/v)): Widely available chemical, which is
used to introduce free amino groups in the epoxy-activated beads and can be
quantified by the 2,4,6-trinitrobenzenesulfonic acid (TNBS) test (see Note 2).
Hazards: Poison, corrosive alkaline solution, causes burns, harmful if swallowed,
inhaled, or absorbed through skin. Toxicity data: LD50 3500 mg/kg oral, rat. Note:
Should be handled in a fume hood with safety glasses and gloves.
4. Ninhydrin (1,2,3-triketohydrindene monohydrate): Widely available chemical that
is light sensitive. Ninhydrin reacts with free amines (2:1 molar ratio) giving a
purple product (Ruhemann’s purple resonance structure). Used as a 0.2% (w/v)
solution in ethanol for the qualitative determination of aliphatic amines on the
agarose beads (see Note 3). Hazards: Harmful if swallowed; skin, eye, and respiratory
irritant. Toxicity data: LD50 78 mg/kg intraperitoneal, mouse. Note: Should
be handled in a fume hood with safety glasses and gloves.
5. Cyanuric chloride (2,4,6-Trichloro-sym-1,3,5-triazine; Chloro-triazine; Trichlorocyanidine):
This is widely available. A high purity (99%) compound should be
used. It is a very reactive compound and must be stored at 2–8°C in an anhydrous
environment. It is recommended to recrystallize in petroleum ether (see Note 4).
Hazards: Poison, lachrymator, and irritant to eyes, skin, and respiratory system.
May be harmful if swallowed. Toxicity data: LD50 485 mg/kg oral, rat. Note:
Should be handled in a fume hood with safety glasses and gloves and treated as a
possible cancer hazard.
6. Amines: For the development of the artificial PpL, the compounds utilized to
sequentially substitute the chlorines of the triazine molecule were: L-alanine