Facile Peptide Macrocyclization and Multifunctionalization via Cyclen Installation

Abstract

Cyclen-peptide bioconjugates are usually prepared in multiple steps that require individual preparation and purification of the cyclic peptide and hydrophilic cyclen derivatives. An efficient strategy is discovered for peptide cyclization and functionalization toward lanthanide probe via three components intermolecular crosslinking on solid-phase peptide synthesis with high conversion yield. Multifunctionality can be conferred by introducing different modular parts or/and metal ions on the cyclen-embedded cyclopeptide. As a proof-of-concept, a luminescent Eu³⁺ complex and a Gd³⁺-based contrasting agent for in vitro optical imaging and in vivo magnetic resonance imaging, respectively, are demonstrated through utilizing this preparation of cyclen-embedded cyclic arginylglycylaspartic acid (RGD) peptide.

Keywords: cyclen‐peptide conjugates; lanthanides; luminescence; solid phase peptide synthesis; αvβ3 integrin.

Figure 1

Schematic representation of the diverse biomedical applications for cyclen‐embedded peptide macrocyclization strategy in radiotherapy, receptor‐specific in vitro luminescence imaging, and fast responding in vivo MRI.

Scheme 1

The synthetic scheme of cyclen‐embedded cyclopeptides cyc‐RGD and cyc‐RGD‐cs constructed on resin‐bound RGD peptide.

Figure 2

a) General structure of Ln‐cyc‐RGD‐cs for visible to NIR luminescent imaging upon different metals coordination. The normalized excitation (in black) and emission spectra (in color) of b) Eu‐cyc‐RGD‐cs (20 µm), c) Sm‐cyc‐RGD‐cs (30 µm), and d) Yb‐cyc‐RGD‐cs (84 µm) in 10 mm HEPES buffer measured at room temperature. e) Immunoluminescence imaging of α _v β ₃ in T24 (α _v β ₃ ⁺), U‐87 MG (α _v β ₃ ⁺) and MRC‐5 (α _v β ₃ ⁻) cells after incubation of 50 µm Eu‐cyc‐RGD‐cs in medium for 24 h (scale bar: 50 µm).

Figure 3

a) General structure of M‐cyc‐RGD and its potential biomedical applications upon different metal coordination. b) HPLC chromatograms of non‐radioactive isotope labeling of M‐cyc‐RGD. c) Stability tests of Gd‐cyc‐RGD and peptide 1 against enzymatic peptide degradation as monitored by HPLC. Peptide remaining (%) was calculated by Δcomplex/ΣΔmixture. d) Relaxation rates 1/T₁ and e) 1/T₂ versus Gd‐cyc‐RGD and Gd‐DOTA concentrations. f) Quantification of T₁ contrast enhancement of Gd‐cyc‐RGD and Gd‐DOTA in tumor over 8 h. g) T₁‐weighted MRI scans of α _v β ₃‐positive U‐87 MG cell tumor xenografts in nude mice treated with 0.1 mmol kg⁻¹ of Gd‐cyc‐RGD or Gd‐DOTA over a period of 8 h. Data are expressed as the mean ± standard deviation (SD) of three independent experiments. ^** p < 0.01 and^*** p < 0.001 as calculated by the Student's t‐test.

Figure 4

The representative conformations of a) linear RGD peptide, b) Y‐cyc‐RGD, and c) Y‐cyc‐RGD‐cs (using Y³⁺ as an analog for Gd³⁺ and Eu³⁺ to avoid spin contamination) with α _v β ₃ protein (blue) over 100 ns of molecular dynamics simulation. The calculated generalized Born (GB) and Poisson‐Boltzmann (PB) values represent the binding free energy.