Recursive deconvolution of a 729-membered peptide library has identified three active sequences, in which both Ser and His are present in one of the two tripeptidic chains generated on a steroidal scaffold (see structural formula), for the cleavage of an activated p-nitrophenyl ester. This combinatorial approach aims at searching for serine-protease-like activity.
A series of peptidosteroid derivatives containing two independent peptide chains in which Ser and His are incorporated were synthesized by solid-phase peptide synthesis. The activity of the different compounds in the hydrolysis of the activated substrate NF31 was assessed in a stepwise fashion. First, the different resin-bound derivatives 6a-l and 6x-z were individually assayed for serine esterification in the absence of water. The use of a colored substrate allowed for a visual identification of the most active compounds. Through the inclusion of control substances, the involvement of histidine in the mechanism for serine acylation was shown. Second, the hydrolysis and methanolysis of the different acylated derivatives 8a-l and 8x were evaluated using UV spectroscopy, again indicating the involvement of histidine. The feasibility of applying the above procedures in a combinatorial context was proven via the screening of artificial libraries, created by mixing the different resin-bound peptidosteroid compounds. In this respect, the use of a photocleavable linker allowed for the unambiguous structural characterization of the selected members via application of single-bead electrospray tandem mass spectrometry.
A convergent synthesis has been developed for the preparation of solid-phase bound construct 1, consisting of an orthogonally protected trifunctional core structure that is attached to TentaGel via a photocleavable linker. [structure: see text]
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