Dynamic covalent self-assembly of mechanically interlocked molecules solely made from peptides

Abstract

Mechanically interlocked molecules (MIMs), such as rotaxanes and catenanes, have captured the attention of chemists both from a synthetic perspective and because of their role as simple prototypes of molecular machines. Although examples exist in nature, most synthetic MIMs are made from artificial building blocks and assembled in organic solvents. The synthesis of MIMs from natural biomolecules remains highly challenging. Here, we report on a synthesis strategy for interlocked molecules solely made from peptides, that is, mechanically interlocked peptides (MIPs). Fully peptidic, cysteine-decorated building blocks were self-assembled in water to generate disulfide-bonded dynamic combinatorial libraries consisting of multiple different rotaxanes, catenanes and daisy chains as well as more exotic structures. Detailed NMR spectroscopy and mass spectrometry characterization of a [2]catenane comprising two peptide macrocycles revealed that this structure has rich conformational dynamics reminiscent of protein folding. Thus, MIPs can serve as a bridge between fully synthetic MIMs and those found in nature.

Figure 1:. Strategy for a dynamic covalent self-assembly synthesis of MIPs.

(a) The mechanically interlocked structure of lasso peptide MccJ25 (L1) with highlighted stopper residues Phe-19 and Tyr-20 (cis/trans configuration of amide bonds is not taken into account). (b) Schematic representation of the synthesis protocol for MIPs made from engineered lasso peptides. After selective substitution of lasso peptide L1 by Cys residues, enzymatic cleavage of a Cys-decorated variant in its loop region yields [2]rotaxane intermediates which undergo dynamic self-assembly into topologically complex MIPs via disulfide bonding. The positioning of Cys residues controls the pathway selection and thus the product distribution in the resulting DCL. A desired MIP can be isolated from the DCL, is stable and can be subsequently modified and functionalized in aqueous solution.

Figure 2:. Synthesized set of Cys-decorated lasso peptide variants.

(a) Sequence of wild-type MccJ25 (L1) with highlighted substitution sites (orange) and predetermined cleavage sites (grey lines) in the loop region. Trypsin cleaves variants with a substitution by Arg-12 between Arg-12/Cys-13 (green rhombus) and thermolysin cleaves between Phe-10/Val-11 (purple circle). The link between Gly-1 and Glu-8 represents the isopeptide bond. (b) Set of engineered MccJ25 variants L2–L8 used in this study. (c) Structure of wild-type MccJ25 (PDB code 1PP5) with highlighted substitution and cleavage sites.

Figure 3:. Dynamic covalent self-assembly synthesis of different MIPs.

(a) Schematic representation of lasso peptide L5, [2]rotaxane 5 and [c2]daisy chain (5-2H)₂. Addition of DTT quantitatively reduces dimer (5-2H)₂ back to building block 5. (b) Electrospray ionization (ESI) mass spectra showing isotopic patterns of L5 (top), 5 (middle) and (5-2H)₂ (bottom) and (c) the corresponding HPLC traces. The most abundant species after 9 days is the head-to-tail dimer (5-2H)₂ with the structure of a [c2]daisy chain (80% yield as determined by HPLC peak area). (d) Synthesis of [3]rotaxane (4-H)₂, left-handed lasso 2-2H, [2]- and [3]catenane 3-2H and (3-2H)₂ and double-lasso macrocycle (7-3H)₂. Yields were determined by integration of HPLC peak areas. HPLC traces can be found in the Supplementary Information (Supplementary Figs 5 and 33). Due to metastability of DCL (7-3H)_n, a yield could not be determined (n.d.). Reaction conditions: (i) trypsin, (NH₄)HCO₃ (50 mM), pH 7.8, air; (ii) thermolysin, Tris (50 mM), CaCl₂ (0.5 mM), pH 8.0, air; (iii) (NH₄)HCO₃ (50 mM), pH 7.8, air. Building blocks were assembled at a peptide concentration of 0.25 mM. DTT, dithiothreitol.

Figure 4:. Mixed DCLs and building block modifications.

(a) Mixed DCL incorporating dithiol building blocks 3 and 5.The hetereodimer 3•5-4H (m/z 1098.7) is the most abundant constituent (see Supplementary Information for additional data). The bar chart displays the relative abundance of head-to-tail homo- and heterodimers in the DCL (3-2H)_n(5-2H)_n based on LC/MS peak area (see Supplementary Fig. 7). (b) Pre-programming of catenane-forming building block 3 by a S18D substitution yields building block 8. At equimolar concentrations, the DCL (8-2H)_n is shifted towards the monomer compared to building block 3 without S18D substitution. Reaction conditions: (NH₄)HCO₃ (50 mM), pH 7.8, air, overall building block concentration of (i) 250 μM and (ii) 100 μM.

Figure 5:. Structural characterization of peptide [2]Catenane 3-2H by NMR spectroscopy and mass spectrometry.

(a) Chemical structure of [2]catenane 3-2H (cis/trans configuration of amide bonds is not taken into account) and schematic representation to indicate the directionality of the rings (arrows point from N- to C-terminus). (b) MS analysis of [2]catenane 3-2H: (top) mass spectrum and (bottom) CID experiment (25 V collision voltage) with mass-selected [(3-2H)+2H]²⁺ ion. After cleavage of a covalent bond, the [2]catenane ion (m/z 1008.0) fragments into its ring subcomponents (m/z 1015.5 and 1200.5). (c) Schematic representation and MS spectra of post-assembly modification of 3-2H by carboxypeptidase, which selectively removes the Phe-10 residue. Reaction conditions: (i) Carboxypeptidase B and carboxypeptidase Y, sodium acetate (50 mM, pH 6.0), 31 h, rt. (d) Partial ¹H NMR and ¹H,¹H TOCSY spectra (800 MHz, 295 K, H₂O/D₂O = 95:5) of [2]catenane 3-2H (1.03 mM) show the overlap of two peak sets. Signals of the minor conformer are marked with an apostrophe. Correlations between the aromatic protons of Tyr-9 and Tyr-20 are highlighted in the TOCSY spectrum. (e) Solution NMR structure of the major co-conformation of [2]catenane 3-2H in water. The backbone of all residues and selected side chains are shown as sticks. Hydrogen and backbone oxygen atoms are removed for the sake of clarity, nitrogen atoms are shaded darker and the disulfide bond is colored yellow. The lowest energy structure of the conformational ensemble is shown. An overlay of the top 20 energy-minimized structures can be found in the Supplementary Information (Supplementary Fig. 44).