Design and Synthesis of a Quenched Fluorogenic Peptide Substrate for Human Cytomegalovirus Proteinase

Abstract

A fluorogenic peptide substrate for HCMV proteinase was synthesized by solid-phase peptide synthesis. The amino acid sequence of this substrate is derived from the maturation cleavage site (M site) of the natural substrate, the assembly protein precursor. The minimum sequence for efficient cleavage requires at least seven residues (P4-P3′). A systematic modification of the peptide substrate was carried out to identify positions suitable for the introduction of the fluorescent donor and the quencher acceptor groups.

Development of a Continuous Fluorescence Assay for Rhinovirus 14 3C Protease Using Synthetic Peptides

“…This approach has several disadvanAntiviral Chemistry & Chemotherapy 8(4) tages tn our experience. For example, the presence of charged amino acids within the peptides could complicate solution coupling chemistry, thus additional protection and deprotection of these side groups would be required in order to keep the charged side chains for enhancement of substrate solubility (Pennington & Thornberry, 1994;Holskin et al, 1995;Handa et al, 1995). Use of automated SPPS methods to synthesize the fluorogenic peptides can minimize these problems.…”

“…Fluorescence assays using the donor!quencher pair Edans/Dabcyl have been described for several viral and non-viral proteases (Matayoshi et al, 1990;Wang et al, 1993;Pennington & Thornberry, 1994;Holskin et al, 1995;Handa et al, 1995). In most cases, Edans and Dabcyl moieties were placed at the Nand C termini via solution chemistry.…”

“…However, high background activitywas observed with the uncleaved substrate, caused by the insufficient collisional quenching by p-nitrophenylalanine. To date, a fluorescence assay based on efficient resonance energy transfer has not been used for HRV 3C protease, although a number of quenched substrates based on this mechanism have been described for several other viral proteases (Matayoshi et al, 1990;Handa et al, 1995;Holskin et al, 1995). The fluorescence donorlquencher pair described in these reports are donor 5-[(2-aminoethyl)amino]naphthalene-l-sulphonic acid (Edans) and quencher 4-(4-dimethylaminophenylazo)benzoic acid (Dabcyl).…”

Design and Synthesis of Fluorogenic Trypsin Peptide Substrates Based on Resonance Energy Transfer

“…2). The conventional method for the attachment of EDANS (20), which couples EDANS to the carboxyl side chain of Asp, results in an enormous decrease in yield caused by secondary structure formation of the peptide chain on the resin and sterical hindrance of the bulky EDANS group. Therefore, we preferred the coupling method described by Pennigton (12) using a solution procedure with EDC/HOBt.…”

Peptidyl Fluoro-Ketones as Proteolytic Enzyme Inhibitors

“…Boehringer Ingelheim proposed [89] a leading compound 46,chosen on the bases of the similarity of its amino acid sequence to that of the M-site of HCMV [90] and of its intrinsic inhibitory activity (IC 50 = 1.8 ± 0.3 µM) against HCMV protease (Fig. (35)).…”