Design, synthesis and evaluation of biomimetic affinity ligands for elastases

Abstract

A low‐molecular‐weight biomimetic affinity ligand selective for binding elastase has been designed and synthesized. The ligand was based on mimicking part of the interaction between a natural inhibitor, turkey ovomucoid inhibitor and elastase, and modelled from the X‐ray crystallographic structure of the enzyme–inhibitor complex. Limited solid‐phase combinatorial chemistry was used to synthesize 12 variants of the lead ligand using the triazine moiety as the scaffold for assembly. The ligand library was screened for its ability to bind elastase and trypsin, and two ligands were studied further. Ligand C4/6 [2‐alanyl‐alanyl‐4‐tryptamino‐6‐(α‐lysyl)‐s‐triazine] was found to bind porcine pancreatic elastase, but not trypsin, with a dissociation constant of 6 × 10−5 M and a binding capacity of 21 mg elastase per ml gel. The adsorbent was used to purify elastase from a crude extract of porcine pancreas. Immobilized ligand C4/5 6 [2‐alanyl‐alanyl‐4‐tyramino‐6‐(α‐lysyl)‐s‐triazine] was similarly chosen for optimal binding of elastase from cod and used to purify the enzyme from a crude extract of cod pyloric caeca. Ligand C4/6 was subsequently synthesized in solution and its structure verified by 1H‐NMR. Copyright © 2000 John Wiley & Sons, Ltd.

Protein stabilization with a dipeptide‐mimic triazine‐scaffolded synthetic affinity ligand

“…The reliability of ligand synthesis chemistry is a key strategic element in ligand screening. To confirm that a hit selected from a solid‐phase library is the expected compound, the common procedure is to synthesize the ligand by conventional organic chemistry, to characterize it and to do a covalent coupling to a solid support (Filippusson et al , ; Palanisamy et al , ; Sproule et al , ; Teng et al , ; Roque et al , ). Therefore, ligand 3′/11 was synthesized following conventional organic chemistry methods (as described in the experimental section).…”

“…Sepharose was derivatized with different degrees of activation by stopping the epoxy reaction after different times: 10, 15, 22, 32, 40, 60, 75, 105 and 840 min. After washing thoroughly each sample with distilled water to remove unreacted epichlorohydrin, the epoxy content was determined according to the procedure described by Filippusson et al (). Epoxy‐activated agarose was aminated with ammonia, according to a protocol adapted from (Roque et al , ), using 1.5 ml of ammonia per gram of moist gel, independently of the epoxy content.…”

A biomimetic Protein G affinity adsorbent: an Ugi ligand for immunoglobulins and Fab fragments based on the third IgG‐binding domain of Protein G

“…It is difficult to analyze and characterize the affinity ligands directly synthesized on the solid phase consisting of 6% crosslinked agarose beads. However, Ugi ligands similar to those immobilized on the resin can be synthesized in solution and analyzed with LC/MS and NMR spectroscopy (Filippusson et al, 2000;Haigh et al, 2009;Morrill et al, 2002), in order to at least characterize the synthetic affinity ligands. The solution-phase synthesis of A2C7I1 was performed using DL-glyceraldehyde as the aldehyde reactant of the Ugi reaction, in order to replace the aldehyde-activated agarose component.…”

A Tailor‐Made “Tag–Receptor” Affinity Pair for the Purification of Fusion Proteins

“…Available structural ligands are usually inexpensive to produce but display low selectivity, and as a consequence the purity of the final protein can be compromised, in particular when using metal chelator ligands, which are more prone to leaching 8. A class of synthetic affinity ligands (“biomimetic ligands”) have been employed in affinity purification of some proteins, such as immunoglobulins,9 glycoproteins,10 prion proteins,11 human recombinant factor VII,12 and elastases 13. These can be rationally designed de novo for a specific target to mimic better the structure and binding of natural biological ligands,7a,b and can combine high selectivity with low production cost.…”