Peptide-based capsules with chirality-controlled functionalized interiors - rational design and amplification from dynamic combinatorial libraries

Abstract

Peptides are commonly perceived as inapplicable components for construction of porous structures. Due to their flexibility the design is difficult and shape persistence of such putative structures is diminished. Notwithstanding these limitations, the advantages of peptides as building blocks are numerous: they are functional and functionalizable, widely available, diverse and biocompatible. We aimed at the construction of discrete porous structures that exploit the inherent functionality of peptides by an approach that is inspired by nature: structural pockets are defined by the backbones of peptides while functionality is introduced by their side chains. In this work peptide ribbons were preorganized on a macrocyclic scaffold using azapeptide-aldehyde reactions. The resulting cavitands with semicarbazone linkers arrange the peptide backbones at positions that are suitable for self-assembly of dimeric capsules by formation of binding motifs that resemble eight-stranded β-barrels. Self-assembly properties and inside/outside positions of the side chains depend crucially on the chirality of peptides. By rational optimization of successive generations of capsules we have found that azapeptides containing three amino acids in a (l, d, d) sequence give well-defined dimeric capsules with side chains inside their cavities. Taking advantage of the reversibility of the reaction of semicarbazone formation we have also employed the dynamic covalent chemistry (DCC) for a combinatorial discovery of capsules that could not be rationally designed. Indeed, the results show that stable capsules with side chains positioned internally can be obtained even for shorter sequences but only for combination peptides of (l, l) and (d, l) chirality. The hybrid (l, l)(d, l) capsule is amplified directly from a reaction mixture containing two different peptides. All capsules gain substantial ordering upon self-assembly, which is manifested by a two orders of magnitude increase of the intensity of CD spectra of capsules compared with non-assembled analogs. Temperature-dependent CD measurements indicate that the capsules remain stable over the entire temperature range tested (20-100 °C). Circular dichroism coupled with TD DFT calculations, DOSY measurements and X-ray crystallography allow for elucidation of the structures in the solid state and in solution and guide their iterative evolution for the current goals.

Fig. 1. Design principles: (a) structural features of natural eight β-barrels (PDB codes 5BVL and ; 1GGL), diameters are between opposite C_α carbons given in Å; (b) model of a cavitand (left) and spatial arrangement of side chains in homo- or heterochiral peptides (right); (c) chemical structures of linkers in different cavitands and comparison of their dimensions (a – distance between the aromatic e₄ carbon and C_α carbon in the first amino acid; b – distance between two opposite C_α carbons).

Fig. 2. (a and b) Two strategies for the synthesis of azapeptides; (c) cavitand and capsule formation.

Fig. 3. (a) X-ray structure of (10a)₂ with an enlarged binding motif (green – encapsulated CHCl₃, cyan – encapsulated MeOH, and yellow – encapsulated phenylalanine side chains); (b) molecular surface and volumes (62 ų and 172 ų) of inner cavities in (10a)₂ calculated with the Chimera program.

Fig. 4. (a and b) Experimental and calculated CD and UV spectra of 10b and 13 (chloroform – top, methanol – bottom, TD DFT B3LYP/6-31g+(d,p), σ = 0.2 eV); (c) ¹H NMR spectrum of (10b)₂ (CDCl₃, 600 MHz, 298 K); (d) modelled structure of the capsule in solution based on 2D NMR spectra (including DOSY and ROESY) composed of two trans-hemispheres of (10b)₂; (e) molecular surface and volume (765 ų) of the inner cavity in (10b)₂ calculated with the Chimera program.

Fig. 5. (a) X-ray structure of (11b)₂ with an enlarged binding motif (green – encapsulated CHCl₃, cyan – encapsulated MeOH, and yellow – encapsulated phenylalanine side chains); (b) ¹H NMR spectrum of (11b)₂ (CDCl₃, 600 MHz, 298 K); (c) modelled structure of the capsule in solution based on 2D NMR spectra (including DOSY and ROESY) composed of two trans-hemispheres of (11b)₂; (d) molecular surface and volumes of inner cavities in (11b)₂ (left – crystal structure, 82 ų and 103 ų; right – model, 1129 ų) calculated with the Chimera program.

Fig. 6. (a) ¹H NMR spectrum of (12a)₂ (CDCl₃, 600 MHz, 298 K); (b) fragment of ROESY spectrum of (12a)₂ (most indicative signals are in frames, CDCl₃, 600 MHz, 298 K) and the binding motif with NOE correlations; (c) modelled structure of the capsule in solution based on 2D NMR spectra (including DOSY and ROESY) composed of two trans-hemispheres of (12a)₂; (d) molecular surface and volume (1098 ų) of the inner cavity in (12a)₂ calculated with the Chimera program.

Fig. 7. (a) ¹H NMR spectrum of 12c (CDCl₃, 600 MHz, 298 K); (b) modelled structure of the cavitand in solution; (c) CD spectra of capsule (12a)₂ and cavitand 12c in chloroform (calculated spectrum of 12c: TD DFT B3LYP/6-31g+(d,p), σ = 0.2 eV).

Fig. 8. (a) Self-sorting reaction; (b) ¹H NMR spectra of (12a)₂ and self-sorting reaction of 8 with rac-7a (CDCl₃, 600 MHz, 298 K).

Fig. 9. (a) ¹H NMR spectra of reactions of 8 with (d, l)-6b and with a mixture of (d, l)-6b and (l, l)-6a (CDCl₃, 600 MHz, 298 K); (b) modelled structure of the capsule (11a)(11b) in solution based on 2D NMR spectra (including DOSY and ROESY) composed of two trans-hemispheres; (c) CD and UV spectra of capsules in chloroform ((11a)₂ is not observed in the NMR spectra because of poor solubility but it is soluble enough to measure the ECD spectrum); (d) molecular surface and volume (535 ų) of the inner cavity in (11a)(11b) calculated with the Chimera program.

Fig. 10. Temperature-dependent ECD spectra of (a) (l, d-11b)₂ and (b) (l, l-11a)(d, l-11b) (in tetrachloroethane, normalized using UV spectra, for UV see the ESI†).