Noncanonical Amino Acids for Hypoxia-Responsive Peptide Self-Assembly and Fluorescence

Abstract

Design of endogenous stimuli-responsive amino acids allows for precisely modulating proteins or peptides under a biological microenvironment and thereby regulating their performance. Herein we report a noncanonical amino acid 2-nitroimidazol-1-yl alanine and explore its functions in creation of the nitroreductase (NTR)-responsive peptide-based supramolecular probes for efficient hypoxia imaging. On the basis of the reduction potential of the nitroimidazole unit, the amino acid was synthesized via the Mitsunobu reaction between 2-nitroimidazole and a serine derivate. We elucidated the relationship between the NTR-responsiveness of the amino acid and the structural feature of peptides involving a series of peptides. This eventually facilitates development of aromatic peptides undergoing NTR-responsive self-assembly by rationally optimizing the sequences. Due to the intrinsic role of 2-nitroimidazole in the fluorescence quench, we created a morphology-transformable supramolecular probe for imaging hypoxic tumor cells based on NTR reduction. We found that the resulting supramolecular probes penetrated into solid tumors, thus allowing for efficient fluorescence imaging of tumor cells in hypoxic regions. Our findings demonstrate development of a readily synthesized and versatile amino acid with exemplified properties in creating fluorescent peptide nanostructures responsive to a biological microenvironment, thus providing a powerful toolkit for synthetic biology and development of novel biomaterials.

Structure‐Activity Studies of Nitroreductase‐Responsive Near‐Infrared Heptamethine Cyanine Fluorescent Probes

“…[16,17] Heptamethine cyanine dyes are commonly employed as the molecular scaffold because they have favorable NIR fluorescence properties. [18][19][20][21][22][23][24] But as the field matures and moves toward clinical translation, there is a need to optimize the molecular design and create NTR responsive probes that have a precise combination of favorable photophysical and pharmaceutical properties. [25] Heptamethine cyanine dyes are inherently hydrophobic molecules, and it is a synthetic challenge to design and prepare them as selective NTR probes with high water solubility, along with rapid enzyme-catalyzed kinetics and a high level of "turn on" NIR fluorescence.…”

“…The research field has progressed from fluorescent probes with visible wavelength emission to probes that absorb and emit NIR light which is known to penetrate further through opaque biological media including skin and tissue [16,17] . Heptamethine cyanine dyes are commonly employed as the molecular scaffold because they have favorable NIR fluorescence properties [18–24] . But as the field matures and moves toward clinical translation, there is a need to optimize the molecular design and create NTR responsive probes that have a precise combination of favorable photophysical and pharmaceutical properties [25]…”

In Vivo Self‐Assembly Induced Cell Membrane Phase Separation for Improved Peptide Drug Internalization

“…In addition, peptides were enriched in the liver,w hich was consistent with other nanodrugs. [25] Second, to observe the drug delivery effect of this strategy in vivo,t he nude mice were pretreated with KYp or KY in the tail vein and then injected with Cy5-labeled KLAK. After 24 h, ex vivo FL imaging was performed on the isolated tumor, and the semiquantitive analysis showed that FL intensity at the tumor…”

Supramolecular Nanomedicines of In-Situ Self-Assembling Peptides

“…Recently, Yu and co-workers designed and synthesized an unconventional amino acid that reacts with NTR (nitro-reductase) to regulate the self-assembly of peptides into a supramolecular probe for morphological transformation, which could be used to efficient hypoxia imaging. (Hu et al, 2021). The nitroimidazole part was integrated into the side chain of alanine, the NTR response amino acid was designed, and the nitro position of the nitroimidazole was optimized to obtain 2-nitroimidazole-1acylalanine with the maximum reduction ability, called A(2NI) (Figure 11A).…”