Dual Stimuli-Responsive Dynamic Covalent Peptide Tags: Toward Sequence-Controlled Release in Tumor-like Microenvironments.

Maksymilian Marek Zegota,Michael Andreas Müller,Svenja Morsbach,Seah Ling Kuan,David Y W Ng,María Martínez-Negro,Pedro M P Gois,Tanja Weil,Jaime A S Coelho,Gönül Kizilsavas,Pierpaolo Moscariello,Bellinda Lantzberg,Manfred Wagner

doi:10.1021/jacs.1c06559

Abstract

Dynamic covalent chemistry (DCvC) has emerged as a versatile synthetic tool for devising stable, stimuli-responsive linkers or conjugates. The interplay of binding affinity, association and dissociation constants exhibits a strong influence on the selectivity of the reaction, the conversion rate, as well as the stability in aqueous solutions. Nevertheless, dynamic covalent interactions often exhibit fast binding and fast dissociation events or vice versa, affecting their conversion rates or stabilities. To overcome the limitation in linker design, we reported herein dual responsive dynamic covalent peptide tags combining a pH responsive boronate ester with fast association and dissociation rates, and a redox-active disulfide with slow formation and dissociation rate. Precoordination by boronic acid–catechol interaction improves self-sorting and selectivity in disulfide formation into heterodimers. The resulting bis-peptide conjugate exhibited improved complex stability in aqueous solution and acidic tumor-like extracellular microenvironment. Furthermore, the conjugate responds to pH changes within the physiological range as well as to redox conditions found inside cancer cells. Such tags hold great promise, through cooperative effects, for controlling the stability of bioconjugates under dilution in aqueous media, as well as designing intelligent pharmaceutics that react to distinct biological stimuli in cells.

Highlights

Stimulus-responsive linker chemistry that can differentiate between the biochemical and physical differences manifested by tumor and normal tissues and respond has emerged as a central tool for the design of smart therapeutics and bioconjugates.[1−3] For example, systems that exploit the acidic extracellular matrix, acidic intracellular endosomes, elevated temperature, and higher glutathione concentrations found in cancer cells have been designed to trigger drug release selectively.[2,4]
An example of Dynamic covalent reactions (DCvR) exhibiting fast kon rates is based on phenylboronic acid chemistry with catechol groups, which proceed with fast kon rates of about 103 M−1 s−1,7,8 and is comparable to one of the fastest known bioorthogonal reactions, i.e., inverse electron demand Diels−Alder.[9]
By exploiting the cooperative effect of a multivalent, fast dynamic covalent reaction of boronate esters with that of a slower and more stable dynamic disulfide formation, we have overcome the limitation of each chemistry and showed that a robust system which is dual responsive with tunable binding affinity can be achieved through rational chemical sequence programming

Summary

■ INTRODUCTION

Stimulus-responsive linker chemistry that can differentiate between the biochemical and physical differences manifested by tumor and normal tissues and respond has emerged as a central tool for the design of smart therapeutics and bioconjugates.[1−3] For example, systems that exploit the acidic extracellular matrix, acidic intracellular endosomes, elevated temperature, and higher glutathione concentrations found in cancer cells have been designed to trigger drug release selectively.[2,4] the field is still fraught with challenges due to unsatisfactory systemic stability or premature drug release of most drug delivery systems.[1,2] In this regard, a fine balance between stability and reversibility, as well as sensitive response to small changes in the environmental parameters, are highly sought after features when designing linkers for biomedical applications. After precoordination based on the boronic acid−catechol interaction, 2Red can be selectively oxidized to 2Ox by forming a disulfide bridge (Figure 5a) We expect that this secondary S−S dynamic covalent bond locks the conjugate as a heterodimer introducing a reductive environment as a new stimulus for dissociation. A solution of 2Ox formed by oxidizing 2Red could be reduced subsequently in situ without prior purification by addition of the reducing agent TCEP in slight excess (2.7 equiv), resulting in nearly quantitative hydrolysis of the bis-peptide into the monomeric peptide sequences (BSCSB and OSCSO) under acidic conditions (Figure 5d). The uptake of DL488 ≃ TAT was observed while the negative control 2, with no disulfide formation, was not internalized (Figure 7c) These results suggest that the linkers are stable due to the cooperative effect of two DCv interactions when applied to the acidic extracellular environment. The linker chemistry can potentially be adopted for more invasive cancer phenotypes

■ CONCLUSION

■ ACKNOWLEDGMENTS

■ REFERENCES