Universal Access to Protease Chemiluminescent Probes through Solid-Phase Synthesis.

Maria Ponomariov,Doron Shabat,Ori Green

doi:10.1021/acs.bioconjchem.1c00384

Maria Ponomariov, Doron Shabat + Show 1 more

Open Access

https://doi.org/10.1021/acs.bioconjchem.1c00384

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Journal: Bioconjugate chemistry	Publication Date: Sep 22, 2021
Citations: 6	License type: CC BY 4.0

Affiliation: Tel Aviv University

Abstract

Protease chemiluminescent probes exhibit extremely high detection sensitivity for monitoring activity of various proteolytic enzymes. However, their synthesis, performed in solution, involves multiple synthetic and purification steps, thereby generating a major limitation for rapid preparation of such probes with diverse substrate scope. To overcome this limitation, we developed a general solid-phase-synthetic approach to prepare chemiluminescent protease probes, by peptide elongation, performed on an immobilized chemiluminescent enol-ether precursor. The enol-ether precursor is immobilized on a 2-chlorotrityl-chloride resin through an acrylic acid substituent by an acid-labile ester linkage. Next, a stepwise elongation of the peptide is performed using standard Fmoc solid-phase peptide synthesis. After cleavage of the peptide-enol-ether precursor from the resin, by hexafluoro-iso-propanol, a simple oxidation of the enol-ether yields the final chemiluminescent dioxetane protease probe. To validate the applicability of the methodology, two chemiluminescent probes were efficiently prepared by solid-phase synthesis with dipeptidyl substrates designed for activation by aminopeptidase and cathepsin-B proteases. A more complex example was demonstrated by the synthesis of a chemiluminescent probe for detection of PSA, which includes a peptidyl substrate of six amino acids. We anticipate that the described methodology would be useful for rapid preparation of chemiluminescent protease probes with vast and diverse peptidyl substrates.

Highlights

Protease chemiluminescent probes exhibit extremely high detection sensitivity for monitoring activity of various proteolytic enzymes
The synthesis of fluorogenic protease probes is usually performed in solution, where the C-terminus of a premade peptidyl substrate is coupled with an amino group of the fluorogenic dye (Figure 1A).[7,8]
solid-phase peptide synthesis (SPPS) can be automated and can be used to create vast libraries of peptidyl substrates, in a method known as Positional Scanning Synthetic Combinatorial Libraries (PS-SCLs).[13,14]

Summary

Introduction

Protease chemiluminescent probes exhibit extremely high detection sensitivity for monitoring activity of various proteolytic enzymes. The synthesis of fluorogenic protease probes is usually performed in solution, where the C-terminus of a premade peptidyl substrate is coupled with an amino group of the fluorogenic dye (Figure 1A).[7,8] This synthetic strategy consumes much time and resources, and requires challenging purification steps, especially when the peptidyl substrate involves the use of protecting groups.[9] In addition, the synthesis in solution cannot be automated or be applied to efficiently prepare combinatorial substrate libraries for fluorescent probes.[3,10] To overcome this limitation, researchers have developed a solid-phase peptide synthesis (SPPS) on a resin with an anchored fluorescent dye.

Results

Conclusion