Synthesis, screening and evaluation of a combined library of tweezer- and tripodal synthetic receptors

The split-mix synthesis of a 6912-member combined library of tweezer- and tripodal synthetic receptors is described. This library was prepared by solid phase attachment of a tweezer hinge, a "locked" tweezer hinge and two triazacyclophane ("TAC") tripodal scaffold, followed by three split-mix cycles using twelve a-amino acid (Gly, Ala, Val, Len, Pro, Phe, Tyr, Lys, Ser, Asp, Gln, His) derivatives. Using fluorescence microscopy and image analysis, the resulting library was screened in aqueous phosphate buffer with fluorescent fragments of the cell wall of Gram-positive Staphylococcus aureus i.e. Ds-Gly-D-Ala-D-Ala-OH and Ds-Gly-D-Ala-D-Lac-OH as well as FITC-labeled peptidoglycan fragments. Decoding of selected beads by Edman degradation gave the structures of the possible synthetic receptors, of which thirteen were resynthesized on the solid phase, including one using a cleavable linker containing resin for confirmation of the quality of the resynthesized receptor. Remarkable binding selectivities were observed, for example the presence of Lys (AA(3)) in almost half of the sequenced receptors arms binding to Ds-Gly-D-Ala-D-Ala-OH, which is less the case in the receptors binding Ds-Gly-D-Ala-D-Lac-OH. Especially prominent was the presence of a Pro residue as AA(3) in more than half of the arms of the sequenced receptors. The observed selectivities were not reflected in the binding constants of representative resynthesized synthetic receptors attached to beads, which were all in the range of 500 M-1 in phosphate buffer. Moreover, this showed that, in contrast to an non-aqueous system, the third arm of the tripod did not contribute to the binding of Ds-Gly-D-Ala-D-Lac-OH, since in chloroform binding constants -also determined on the beads- were observed of 11,700 M-1 and 5,400 M-1 for a tripod and tweezer receptor, respectively.