A Facile Cyclization Method Improves Peptide Serum Stability and Confers Intrinsic Fluorescence

Abstract

Peptides are a growing class of macromolecules used in pharmaceutics. The path toward the clinical use of candidate peptides involves sequence optimization and cyclization for stability and affinity. For internalized peptides, tagging is also often required for intracellular trafficking studies, although fluorophore conjugation has an impact on peptide binding, permeability, and localization. Herein, a strategy based on cysteine arylation with tetrafluoroterephthalonitrile (4F-2CN), which simultaneously cyclizes peptides and imparts fluorescence, is reported. The 4F-2CN cyclization of an M2 macrophage-targeting peptide yields, in a single step, a peptide with improved serum stability, intrinsic fluorescence, and increased binding affinity. In a murine breast cancer model, it is demonstrated that the intrinsic fluorescence from the cyclized peptide is sufficient for monitoring biodistribution by whole-organ fluorescence imaging and cell internalization by flow cytometry.

Keywords: cyclization; fluorescence; imaging agents; peptides; pharmaceutics.

Figure 1

(A) Absorption spectra of peptide 1 and 2. Magnification on the region of interest (Right insert). Fluorescence emissions of the peptides at excitation wavelength of (B) 420 nm and (C) 350 nm.

Figure 2

(A) Amino acid sequences of AcM2pep(RY) (Peptide 3). (B) Binding study with M1 and M2 macrophages detected with streptavidin-Alexa Fluor 647. (C) Serum stability of peptide 1 and 3. * denotes peak of the intact peptide (D) Confocal images of untreated (left) and peptide 1-treated (right) M2 macrophages.

Figure 3

(A) Xenogen images of perfused organs from tumor-bearing mice injected with PBS (left) and peptide 1 (right). (B) Quantification of the fluorescence signal in each organ. * denotes statistical significance based on unpaired Student’s t test (P < 0.05) (C) Intratumoral distribution of peptide in CD11b⁺ (macrophage) and CD11b⁻ (non-macrophage) populations. * denotes statistical significance based on unpaired Student’s t test (P < 0.05) (D) Representative 4T1 tumor sections from PBS-treated (top) and peptide 1-treated (bottom) groups. Green: peptide, Red: F4/80 macrophage stain, Blue: DAPI.

Scheme 1

4F-2CN cyclization of M2pep(RY) with N-terminal Cys (Peptide 1) and Hcy (Peptide 2).