A 3D Peptide/[60]Fullerene Hybrid for Multivalent Recognition

Abstract

Fully substituted peptide/[60]fullerene hexakis-adducts offer an excellent opportunity for multivalent protein recognition. In contrast to monofunctionalized fullerene hybrids, peptide/[60]fullerene hexakis-adducts display multiple copies of a peptide in close spatial proximity and in the three dimensions of space. High affinity peptide binders for almost any target can be currently identified by in vitro evolution techniques, often providing synthetically simpler alternatives to natural ligands. However, despite the potential of peptide/[60]fullerene hexakis-adducts, these promising conjugates have not been reported to date. Here we present a synthetic strategy for the construction of 3D multivalent hybrids that are able to bind with high affinity the E-selectin. The here synthesized fully substituted peptide/[60]fullerene hybrids and their multivalent recognition of natural receptors constitute a proof of principle for their future application as functional biocompatible materials.

Keywords: Fullerenes; Glycomimetic; Lectin; Multivalency; Peptides.

Figure 1

Different steps in the molecular scaffold construction: a) In vitro identification of a mimetic (IELLQAR) of a natural ligand (sLe^X ); b) Development of a highly substituted peptide/[60]fullerene hybrid; c) Multivalent and selective recognition of E‐selectin (dark‐teal receptors) by the fullerene hybrids, with the potential application of blocking the attachment of sLe^X ‐overexpressing circulating tumor cells (gray cells) to the activated endothelium (pink cells) with the subsequent inhibition of metastasis.

Figure 2

Synthetic analysis of these peptide/[60]fullerene platforms. SPAAC reaction between peptides modified with an azide group: the IELLQARK (active peptide) and ISELRSE (negative control peptide) and the cyclooctyne groups of C₆₀ fullerene hexakis‐adducts to give rise to the symmetric hybrids: the active F₁ (C₆₀‐(IELLQARK)₁₂) and the control F_C (C₆₀‐(ISELRSE)₁₂), as well as the asymmetric Rhodamine B fullerene adducts: the active F₁ ^RB (RB‐C₆₀‐(IELLQARK)₁₀) and control F_C ^RB (RB‐C₆₀‐(ISELRSE)₁₀).

Figure 3

Peptide IELLQARK‐(PEG)₄‐N₃ (P₁ ) was prepared by adding a new lysine amino acid previously modified with the corresponding oligoethylene glycol linker (2) at the C‐terminus in the beginning of the synthesis. Peptide N₃‐(PEG)₄‐IELLQAR (P₂ ) was prepared by incorporating the polyethylene glycol linker (1) at the N‐terminus at the end of the solid phase synthesis (see section 3 in the Supporting Information).

Figure 4

Synthesis of peptide/[60]fullerenes F₁ and F_C via SPAAC reaction between hexakis‐adduct 3 and peptide azides P₁ and P_C , respectively. Reagents and conditions: 3 (1 equiv), P₁ or P_C (12 equiv), DMSO, MW 50 °C, 30 min (for F₁ from P₁ and for F_C from P_C , quant.).

Figure 5

Synthesis of labelled peptide/[60]fullerenes F₁ ^RB and F_C ^RB . Reagents and conditions: i) NaN₃, DMF, 60 °C, 3 days (quant.); ii) EDC⋅HCl, DMAP, CH₂Cl₂/DMF, r.t., overnight (quant.); iii) 6 (1 equiv), 8 (2 equiv), DMSO, MW 50 °C, 30 min (99 %); iv) 9 (1 equiv), 10 (15 equiv), DCC, DPTS, CH₂Cl₂/DMF, r.t., overnight (99 %); v) 11 (1 equiv), P₁ or P_C (10 equiv), DMSO, MW 50 °C, 30 min (for F₁ ^RB from P₁ , 99 %, and for F_C ^RB from P_C , quant.).

Figure 6

SPR sensorgrams of hybrid fullerenes a) F₁ , and b) F₁ ^RB at (12.5–0.78) μM binding to E‐selectin functionalized surfaces (representative graphics for low density (LD) functionalization, see Supporting Information). The association times below 50 s (yellow lines) and dissociation times longer than 600 s (red lines) used for constant determination (Table 1, Supporting Information) are shown.

Figure 7

Cellular assay. a) Confocal microscopy images of HUVEC cells (top row) or TNFα‐treated HUVEC cells (bottom row) incubated with 10 μM of F₁ ^RB (left column) or F_C ^RB (right column). Scale bar: 25 μm. b) Flow cytometry assay of HUVEC cells incubated with 10 μM of F₁ ^RB or F_C ^RB , pretreated (gray checkered bars) or not (smooth gray bars) with 10 ng mL⁻¹ of TNFα. Data presented as mean of the median fluorescence intensity of three replicates±standard deviation.