Structurally disfavoured pseudopeptidic macrocycles through anion templation

Abstract

An anionic dicarboxylate is able to template the formation of geometrically disfavoured macrocycles from a dynamic covalent mixture of open chain oligoimines.

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Peptide-like macrocycles are increasingly interesting molecules with important applications in many different fields.¹ However, their synthesis is usually hampered by the macrocyclization step,² which requires the reaction intermediate to adopt a pre-cyclic conformation. This effective preorganization is often obtained by an inherent folding of the linear precursors,³ or through the implementation of well defined non-covalent interactions.⁴ In this regard a fundamental question arises: can a system with the wrong folding to cyclize be forced to a productive cyclic intermediate? This possibility would give access to geometrically disfavoured macrocycles. For addressing that issue, we have used the conceptual approach of constitutional dynamic chemistry.⁵ The dynamic systems thus generated can self-correct in the presence of external stimuli,^5,6 out of which we selected an anion template⁷ due to their well established interactions with peptidic N–H groups.⁸

In previous studies,⁹ we found that the condensation between pseudopeptidic bis(amidoamines) (1–3, Scheme 1) and aromatic dialdehydes (4) led to the formation of a dynamic mixture of open chain and cyclic oligoimines.¹⁰ When the aliphatic spacer of the bis(amidoamine) was a flexible chain (1), a complicated mixture of compounds was obtained.^9b,c However, when a rigid cyclic spacer was used (2), the formation of the [2+2] macrocycles was strictly controlled by a match/mismatch relationship between the stereocentres of the molecule: the (S,R,R,S)-compounds (2, match) led to the spontaneous macrocyclization, whereas the (R,R,R,R) stereoisomers (3a and b, mismatch) were totally inefficient.^9a,d Consequently, the compounds 3a and b contain the wrong chemical information for an efficient macrocyclization, being ideal for assaying the effect of anion templation on structurally disfavoured dynamic covalent systems, as a proof of concept.

Scheme 1

We initially studied the Phe derivative (3a), according to previous results in a related anion templation process.^9b,c The ¹H NMR spectrum of the reaction between 3a and terephthaldehyde 4 (500 MHz, 23 °C, 1 ∶ 1 CDCl₃ ∶ CD₃OD) after 48 h of equilibration in the absence and in the presence of template 5 displayed remarkable differences.‡ The use of 5 as a template was suggested by molecular modelling studies and was in accordance with our previous experience.^9b,c The non-templated reaction showed the presence of several aldehyde (δ = 10.0–10.2 ppm) and hemiacetal-type protons (δ = 5.6 ppm), implying that the major components of the mixture were open chain oligomers and/or remaining reagent. Remarkably, these signals almost disappeared when the reaction was carried out in the presence of the terephthalate dianion (as the bis-TBA salt, 5), suggesting the formation of cyclic imine products.

Although the NMR spectrum displayed a complicated pattern of broad signals, after performing 2D NMR experiments‡, we concluded that the mixture was composed of two major species in a ∼3.5 ∶ 1 relative proportion. Since neither of them showed aldehyde or acetal type protons, they should be macrocyclic oligoimines of different sizes. Accordingly, we decided to study the reaction mixtures with DOSY, which has been recently used to characterize species of different sizes from a dynamic mixture in situ.¹¹ The DOSY spectrum of the equilibrium mixture of 3a + 4 in the absence of template‡ showed two main groups of signals differentiated in the diffusion scale (and thus, corresponding to species with different molecular sizes). The ones containing exclusively aromatic, aldehyde and X–CH–O protons diffuse at ∼1.50 × 10⁻⁵ cm² s⁻¹, which is a slightly smaller value than the one observed for the TMS used as internal reference in the same sample (1.67 × 10⁻⁵ cm² s⁻¹). This allowed us to assign these signals to the remaining terephthaldehyde and the corresponding mono hemiacetal obtained upon the addition of one CD₃OD molecule. The other signals containing protons from the pseudopeptidic and the phenylene moieties diffuse at a much lower value of D = 3.75 × 10⁻⁶ cm² s⁻¹, implying a considerably larger molecular volume of the implicated species. Since at least two aldehyde proton signals (δ = 10.06 and 10.05 ppm) diffuse at this value, they must correspond to open chain oligomers of [3a + 4]. On the other hand, the DOSY spectrum of the reaction in the presence of 5 showed two main species with very different diffusion rates: one at 6.58 × 10⁻⁶ cm² s⁻¹, corresponding to the major species, and another at 3.82 × 10⁻⁶ cm² s⁻¹ which is the more symmetrical minor compound. Considering the self-diffusion rate of the TMS in the same sample and the corresponding molecular volumes, the major species must be the supramolecular complex formed by the [2+2] tetraimine macrocycle and the template.‡ Accordingly, the other minor compound should be a larger macrocyclic oligomer.§ The reaction mixture was additionally studied by ESI-MS in the anion detection mode.† The main supramolecular species were those formed by the [2+2] macrocyclic tetraimine and the template either alone (at m/z = 588.3, doubly charged) or with one TBA cation (at m/z = 1419.8, singly charged). Other minor peaks sourcing from the corresponding macrocycle–dianion complexes of the [3+3] (m/z = 841.9, doubly charged) or the [4+4] (m/z = 1095.0, doubly charged)¶imines were also detected, although with much lower intensity. These observations were consistent with the NMR data and supported that the anion was able to efficiently induce the formation of the [2+2] macrocycle from the dynamic mixture of linear oligomers, otherwise very disfavoured to undergo macrocyclization. We performed additional experiments to fully understand the dynamic process.‡ The final ¹H NMR spectra were essentially identical either doing the condensation between 3a and 4 in the presence of 5, or by pre-equilibrating the mixture (3a + 4) and then adding 5 to re-equilibrate (Scheme 1).‡ Moreover, we carried out the reaction using different proportions of template (5) with respect to the reactants (3a + 4). According to our expectations, the optimal proportion was 1 ∶ 1 ∶ 0.5 for a 3a ∶  4 ∶  5 ratio. When using a slightly smaller molar ratio of template (0.3), open chain oligomers were readily observed by NMR even after several weeks.‡ These data support the formation of the ([2+2] · template) supramolecular complex as the most stable product, operating under thermodynamic control.

The dynamic process within the templated reaction mixture can be frozen by the in situreduction with NaBH₄ (Scheme 1). In this way, we obtained the tetraamine macrocyclic dimer (6a, 50%), the corresponding trimer (7a) also being isolated as a side product (15%). These are very good overall yields considering the number of independent C–N bonds formed in a one-pot two-step process, and after a careful purification, which is usually a limiting factor for the isolation of this type of compounds. Besides, it is worth mentioning that the corresponding pseudopeptidic building block is structurally disfavoured for macrocyclization. The [2+2] macrocyclic structure was further supported by X-ray diffraction analysis of single crystals of 6a (as its HClO₄ salt, Fig. 1).‡ Regarding the macrocycle, up to four different conformations were found in the solid state. Superposition of them‡ showed that they mainly differ in the disposition of the amino acid side chains and in the conformation of p-phenylene aromatic group. However, they displayed a similar shape of the whole macrocycle and an almost identical conformation for the cyclohexanebis(amide) moieties. Despite that the all-R configurations of the chiral centers of the molecule would render an average D₂ symmetry, the structure lacks symmetry in the solid state. The whole molecule is bent, locating both cyclohexane rings out of the macrocyclic main plane. The most remarkable geometrical feature of the crystal structure of 6a is the conformation of the cyclohexanebis(amide) moieties.¹² Every fragment sets one amide with the N–H bond eclipsed with the vicinal methyne proton of the cyclohexane asymmetric center (N–H/C*–H in cis disposition, Fig. 1, with a torsion angle ranging between 0.13° and 10.15° for the different conformers found in the crystal cell). On a simplified model, we have estimated that this cis disposition would produce a destabilization effect of about 6.4 kcal mol⁻¹ per eclipsed unit.‡ This arrangement is repeated along the macrocyclic structure, leading to alternating cis/trans/cis/trans relative conformation of N–H/C*–H groupings. This uncommon geometry sets the consecutive amide N–H protons of every pseudopeptidic moiety in syn, and all of them pointing to the same face of the macrocyclic ring (highlighted in yellow in Fig. 1). Overall, the crystal structure reflected the disfavoured geometry of macrocycle 6a, with a large disorder and a strained conformation. This is also in agreement with the NMR data of 6a as the free amine in solution.‡

Fig. 1 Top and side views of one conformation observed in the crystal structure of [6a·4HClO₄]. The other geometries are given in the ESI.‡

We performed molecular modelling calculations (Fig. 2A–C) to visualize the effect of the terephthalate dianion on the hypothetical [2+2] aminoaldehyde intermediate, previous to the formation of the fourth imine bond. In the absence of the template, the linear molecule located both ends at a long distance (8.4 Å) with both cyclohexanebis(amide) moieties in anti (Fig. 2A). In the presence of the template (Fig. 2B) the H-bonds between the carboxylate groups and the amide protons forced two alternated amide groups to rotate ∼180°, setting the corresponding N–H moieties in syn‡, as observed in the crystal structure of 6a. This conformational rearrangement locates both ends of the linear molecule at the appropriate distance (3.7 Å) and geometry for the C=N bond formation (Fig. 2B). Actually, the supramolecular complex formed by the [2+2] macrocyclic tetraimine and the dianion template displays a very good geometrical complementarity, retaining the four H-bonds of the syn-amides and with the aromatic diimines fully conjugated in a s-trans conformation (Fig. 2C). We also modeled the [3+3] macrocyclic supramolecular complex which led to the observed side product 7a (Fig. 2D). The optimized geometry showed two pseudopeptidic moieties with four syn-amide N–H conformations and the third bis(amide) with two N–H in anti. Besides, the four N–H groups in syn form the “binding site” for the template. Therefore, the formation of the [3+3] macrocycle can be explained as a result of an incomplete reorganization, where one of the three building blocks has not suffered the necessary conformational rearrangement to interact with the template.

Fig. 2 Aminoaldehyde intermediate in the absence (A) and in the presence (B) of the dianionic template. Minimized geometries for the complexes of the (C) [2+2] and (D) [3+3] macrocyclic oligoimines and the template (non-polar H atoms omitted in A–D).

Attracted by this hypothesis, we reasoned that an anion templated dynamic system subjected to a kinetic trap before the equilibration process is fully achieved would lead to an increased amount of larger macrocycles. That was exactly what we observed when using the pseudopeptidic bis(amide) bearing iPr side chains (3b). Thus, when performing the same templated reaction with the corresponding Val derivative, a precipitate was readily formed after a few hours (<8 h, a shorter time than the one needed to reach the equilibrium). The NaBH₄ reduction of this suspension yielded the corresponding trimer (7b, 40%) and tetramer (8b, 20%) after careful purification, the [2+2] macrocycle (6b) being detected just as a trace product. Accordingly, as suggested by the modelling studies of the trimer with 3a, the larger oligomers are produced by an incomplete structural reorganization of the building blocks. Thus, the precipitation observed with 3b should kinetically trap the larger macrocycles initially formed.|| However, for 3a, the dynamic system remains in solution, which allows the reconstitution by re-equilibration to yield the best fitted anion–macrocycle complex.**

In summary, we have shown how an anion template can rule the outcome from a dynamic covalent mixture of pseudopeptidic oligoimines. Despite that the system is structurally much disfavoured for the cyclization, the interactions with the anionic template force the correct folding to yield the best fitted macrocycle. Moreover, the fine tuning of the thermodynamic and kinetic parameters permitted to control the size of the final macrocycles. Very importantly, the correct folding for the macrocyclization implies that the pseudopeptidic moiety has to adopt an energetically disfavoured conformation. From the molecular evolution perspective, this minimalistic system represents an outstanding example of dynamic correction, which demonstrates the power of supramolecular chemistry and, especially, anion templation for the preparation of structurally disfavoured molecular architectures.

This work was supported by the Spanish Ministerio de Ciencia e Innovación (CTQ2009-14366-C02).

Notes and references

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Footnotes

† This article is part of the ‘Emerging Investigators’ themed issue for ChemComm.

‡ Electronic supplementary information (ESI) available: Further details regarding the synthesis, NMR, X-ray or modelling studies. CCDC 765248. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c0cc01784a

§ Due to statistical reasons, we anticipated that this species should correspond to the [3+3] macrocycle, which was confirmed after the reduction and purification of the reaction mixture, see below.

¶ Since the tetramer was not detected after the reduction of the imine functions, we believe that it was formed in the ESI-MS conditions. Another explanation of this mass peak could source from a non-covalent cluster of two molecules of the dimer with one dianion template.

|| The observed aggregation (precipitation) could also affect the equilibria of the dynamic system in the case of 3b (see ESI‡).

** Larger macrocycles were also formed when we reduced the templated reaction with 3a before the equilibrium was reached (see ESI†).

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