General dual functionalisation of biomacromolecules via a cysteine bridging strategy.

Stephen J Walsh,Hannah F Sore,Jeremy S Parker,Hikaru Seki,Jonathan D Bargh,Jason S Carroll,Jessica Iegre,David R Spring

doi:10.1039/d0ob00907e

Abstract

Site-selective modification of peptides and proteins has resulted in the development of a host of novel tools for the study of cellular systems or the synthesis of enhanced biotherapeutics. There is a need for useful methodologies that enable site-selective modification of native peptides or proteins, which is even more prevalent when modification of the biomolecule with multiple payloads is desired. Herein, we report the development of a novel dual functional divinylpyrimidine (dfDVP) platform that enables robust and modular modification of peptides, antibody fragments and antibodies. These biomacromolecules could be easily functionalised with a range of functional payloads (e.g. fluorescent dyes, cytotoxic warheads or cell-penetrating tags). Importantly, the dual functionalised peptides and antibodies demonstrated exquisite bioactivity in a range of in vitro cellular assays, showcasing the enhanced utility of these bioactive conjugates.

Highlights

The modification of proteins can be leveraged for a variety of tasks such as the study of cellular processes or the generation of bioconjugate therapeutics.[1,2] To an even greater extent, dual modification of peptides or proteins can provide valuable tools to aid in these investigations.[3,4] Incorporation of multiple payloads can take on many formats; such as attachment of Förster resonance energy transfer (FRET) pairs, half-life extension strategies of protein/peptide-drug conjugates, increased cellular permeability of peptide-based drugs/imaging agents or macromolecular theranostics
We have previously reported the used of divinylpyrimidine (DVP) reagents for the site-selective synthesis of exceptionally stable antibody–drug conjugates (ADCs), and divinyltriazine reagents to synthesise functionalised macrocyclic peptides.[34,35]
The dual functional divinylpyrimidine (dfDVP) reagent was designed to contain a fluorescent dye, from reaction with fluorescein isothiocyanate (FITC) and an alkyne-containing amino acid, both of which could be inserted during on-resin synthesis

Summary

Introduction

The modification of proteins can be leveraged for a variety of tasks such as the study of cellular processes or the generation of bioconjugate therapeutics.[1,2] To an even greater extent, dual modification of peptides or proteins can provide valuable tools to aid in these investigations.[3,4] Incorporation of multiple payloads can take on many formats; such as attachment of Förster resonance energy transfer (FRET) pairs, half-life extension strategies of protein/peptide-drug conjugates, increased cellular permeability of peptide-based drugs/imaging agents or macromolecular theranostics. Dual modification can typically be achieved in two ways: (1) modification of two different amino acids with separate payloads, or (2) use of a single linker with orthogonal reactive handles to introduce the distinct functionality.[5] While this can be relatively straightforward in short peptides, site-selective dual modification of large proteins can be challenging.[6]. Linear peptides typically suffer from poor circulatory stability and cell permeability, limiting their therapeutic potential.[7,8] The use of a linker to macrocyclise the peptide can improve these characteristics and the overall pharmacological profile of the peptide drug. The linker can be used to modify the physical properties of the peptide (e.g. solubility, permeability) or can be used to attach functional payloads (e.g. drugs, fluorescent dyes).[9,10] Peptide macrocyclisation linkers that facilitate dual modification with these tags would enable further development of peptide drugs and probes

Objectives

Results

Conclusion