Chiral, sequence-definable foldamer-derived macrocycles

Nature's oligomeric macromolecules have been a long-standing source of inspiration for chemists producing foldamers. Natural systems are frequently conformationally stabilised by macrocyclisation, yet this approach has been rarely adopted in the field of foldamer chemistry. Here we present a new class of chiral cyclic trimers and tetramers formed by macrocyclisation of open-chain foldamer precursors. Symmetrical products are obtained via a [2 + 2] self-assembly approach, while full sequence control is demonstrated through linear synthesis and cyclisation of an unsymmetrical trimer. Structural characterisation is achieved through a combined X-ray and DFT approach, which indicates the tetramers adopt a near-planar conformation, while the trimers adopt a shallow bowl-like shape. Finally, a proof-of-concept experiment is conducted to demonstrate the macrocycles' capacity for cation binding.


Introduction
Nature frequently exploits macrocycles as functional molecules within living systems, 1-3 and many of these and their derivatives have been exploited as therapeutics. 4-6 Some of the most important drugs for human health are naturally-occurring macrocycles, including vancomycin and cyclosporin. The relative success of macrocyclic peptide drugs can be attributed to several factors including stability to degradation, cyclic constraint favouring the active conformation, and high membrane permeability. Inspired by macrocycles' use in Nature and potency in the clinic, chemists have developed numerous cyclisation strategies to stabilise the conformation of small bioactive peptides, including helix "stapling" 7 and cross-linking to stabilise bhairpin structures. 8 Peptides have also been replaced entirely, with articial folded molecules dubbed "foldamers". 9 Entirely abiotic macrocycles have also been used in applications outside biology, particularly in host-guest chemistry. 10 Here, the preorganisation of functionality within the macrocycle enables large binding constants and high guest-specicity.

Synthesis of macrocycles
We have previously described a foldamer architecture where dipolar repulsion between adjacent pyrimidine and imidazolidin-2-one components leads to structures with a turn per monomer of $86 . 15 The attempted synthesis of related pyridine-linked macrocycles by Meth-Cohn failed entirely, generating only oligomeric material, 42 but we hypothesised that the dipole-mediated pre-organisation in our putative linear precursor could favour macrocyclisation over oligomerisation. Similar preorganisation towards macrocyclisation has previously been exploited by Gong in the self-assembly of oligobenzamide foldamers. 26,43 We tested this hypothesis by rst synthesising linear dimer 2a by iterative deprotection/Buchwald-Hartwig coupling steps from a chiral, amino alcoholderived monomeric precursor (see ESI † for procedures). Removal of the N-tert-butyl protecting group on 1a under acidic conditions afforded dimer 2a in 81% yield. Both the N-terminally protected and tert-butyl deprotected intermediates 1a and 2a were crystalline solids, with the latter puried conveniently by trituration with hot hexanes.
Examination of their single crystal X-ray structures revealed that the expected dipole-opposed conformation was adopted, giving both molecules an overall crescent shape (Scheme 1B). We were therefore condent that treatment of dimer 2a under Buchwald-Hartwig cross-coupling conditions would lead directly to the C 2 -symmetrical macrocycle 3a, since the initial tetrameric product of homo-coupling would be pre-organised via dipolar repulsion for a second coupling reaction to generate the macrocycle. Thus, treatment of 2a with Pd 2 (dba) 3 , Xantphos and Cs 2 CO 3 under reux in toluene afforded macrocycle 3a in 56% isolated yield, with no evidence of the formation of larger macrocycles or polymeric material. With this synthetic strategy validated we proceeded to synthesise a further three dimeric precursor molecules 2b-d. 44 Upon treatment under crosscoupling conditions all were converted to the corresponding C 4 -symmetrical macrocycles 3b-d in isolated yields of 61-85%.
relative to the tetrameric homologues, with 5a and 5b obtained in 6% and 29% yields respectively. Macrocycle 5b is especially noteworthy as it is entirely "sequence-dened", with the cyclic ordering of the monomers predetermined by the order of monomer addition in the preparation of the linear precursor.

Conformational studies
In an effort to understand its conformational behavior, single crystals of macrocycle 3a were grown by vapour diffusion (Fig. 2A). Attempts to generate diffraction-quality crystals of the remaining macrocycles were unsuccessful. As expected, all four sidechains of 3a are projected from a single face of the macrocycle in a highly controlled manner. Inspection of the crystal packing reveals a layered, back-to-back stacking arrangement between planes of macrocycles ( Fig. 2B-D), with the relative orientation of macrocycles between layers controlled in part by a dipole-opposed arrangement between adjacent imidazolidin-2-ones (Fig. 2C). Due to disorder and weak diffraction the Xray data were insufficient to gain further structural insights, so we proceeded to explore the macrocycles' conformational behavior by DFT (Fig. 3).
The lowest energy conformation of the tetramer was found to be a shallow bowl, with all C]O groups puckered outwards from the same face of the macrocycle, and a transverse O-O distance of 5.2Å. Two low energy conformers were identied for the trimer, corresponding to a macromolecular "ring-ip", in which the sidechain substituents are placed in pseudo-axial or pseudo-equatorial positions (Fig. 3B). The equatorial conformer is lower in energy by 5.8 kcal mol À1 and represents the global energy minimum. No "mixed" conformers (in which one imidazolidin-2-one is puckered in an opposing direction to the others) are identied as minima. The less-planar structure of the trimer relative to the tetramer is also supported by 1 H NMR data: the inward-pointing pyrimidine hydrogens in tetramers 3a-3d appear far downeld ($9.9 ppm), likely due to the deshielding effect of the proximal oxygen lone-pairs, whereas the equivalent peaks in the trimers 5a and 5b appear at $8.7 ppm indicating that these hydrogens are subject to the deshielding effect of the lone pairs to a much lesser degree.
Guest binding. The structural and computational data were indicative of the tetrameric macrocycles possessing a central pore with the four urea carbonyl groups directed towards its centre. We anticipated that cations would be well-stabilised within the macrocycle, as had already been observed with Cs + during the isolation of 3a. We were particularly interested in the binding of ammonium cations since methylated lysines form part of the epigenetic histone code, and selective methods to probe them are therefore of importance. [45][46][47] The binding of macrocycle 3d to hexadecyltrimethylammonium chloride was examined by 1 H NMR titration in CDCl 3 (Fig. 4). 48 Upon treatment with the ammonium salt, H B displays a small downeld shi while H A displays a larger upeld shi. The greater  magnitude shi of H A is consistent with our assumption that binding occurs to the inner edge of the macrocycle, in closer proximity to H A than H B , likely in the manner of "perching" complexes described by Cram, and mediated by CH/O hydrogen bonding between the ammonium a-C-H bonds and imidazolidin-2-one carbonyl groups. [49][50][51] The data are suggestive of 1:2 host:guest complex formation, with tted equilibrium constants of K 11 ¼ 1550 M À1 and K 12 ¼ 28 M À1 . The ability to alter the size and side-chains of the macrocycle raises the prospect of tailoring the selectivity of this cation binding to the desired guest in future studies.
Lastly, an attempt was made to achieve diasteroselective recognition of a chiral guest using dibenzoyl tartaric acid, chosen for its solubility in CDCl 3 , availability in both enantiomeric forms, and ability to form hydrogen bonds to the acceptor-rich environment of the macrocycle interior. However, no difference in binding was observed between enantiomers (see ESI †). We attribute this to the ability of guests to bind the unsubstituted face of the macrocycles, where little stereodifferentiation is feasible; studies are ongoing to synthesise macrocycles incorporating substituents on both faces to address this issue, either by use of C 2 -symmetrical 1,2-disubstituted diamines, or through the use of alternating (R)-and (S)-congured imidazolidine-2-ones in adjacent monomers.

Conclusions
To summarise, we have synthesised and examined the properties of six chiral foldamer-derived macrocycles. The strategy enables their synthesis in entirely sequence-dened manner when required, through synthesis of a linear precursor, or in a convenient semi-dened manner through generation of linear dimers which undergo spontaneous cyclisation upon coupling to form the tetramer. DFT indicates the trimeric macrocycles adopt a bowl-like shape with substituents in a pseudo-equatorial position, while tetramers adopt a planar conformation, as demonstrated by single crystal X-ray diffraction, and bind metal and ammonium cations. The high level of conformational control means these macrocycles are an excellent platform for the controlled positioning of sidechain groups. Current work is ongoing to examine applications of the macrocycles in molecular recognition and catalysis, and to develop a solid-supported second-generation synthetic approach.

Conflicts of interest
There are no conicts to declare.