Design and synthesis of conformationally homogeneous pseudo cyclic peptides through amino acid insertion: investigations on their self assembly

Abstract

Macrocyclic C₂ symmetric peptides, containing bis furanoid triazole amino acids as di-β-peptides linked to a D-α-amino acid or a β-amino acid in each half, have been designed and synthesized. The D-α-amino acid derived product undergoes parallel homo-stacking in solution via amide NH and amide carbonyl oxygen H-bonding; such macrocycles may be used as model systems for artificial ion channels as their unidirectional assembly pattern attributes them with large dipole moments. In contrast, the β-amino acid based compound forms only a conformationally homogeneous cyclic peptide without undergoing self assembly.

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Construction of macrocyclic peptides via H-bonded organic tubular assemblies has become an important area of research for biomimetic materials with potentially useful applications.¹ The desirable prerequisite for such endeavour is the ability to tailor pore sizes and pore surfaces for each specific application. Self-assembled peptide nanotubes (SPNs) constitute a class of materials that allow easy control of pore size.² Modification of the interior property as well as polarity of the SPN can be accomplished through peptide backbone alteration by insertion of amino acids or by introduction of heterocycles.³ It is well recognised that cyclic D-,L-α-peptides with an even number of alternating D- and L-α-amino acids can adopt flat, ring-shaped conformations with the backbone amide functionalities oriented perpendicular to the side chains and the plane of the ring.⁴

Replacement of amide in these macrocycles by triazole leads to a modified macrodipole which is the basic requirement for the construction of synthetic artificial ion channels.^5,6 1,4-Substituted-1,2,3-triazole, an amide bond surrogate (in terms of planarity, polarity, as well as hydrogen bond donating and accepting ability),⁷ is an attractive modification for building up not only cyclic-β-tetrapeptides but also several classes of backbone modified cyclic pseudo-peptides as well as peptidomimetic macrocycles.⁶ The shortest triazole/amide cyclic oligomer having uniform backbone chirality possesses a small macrodipole because of the alternative orientation of functional groups. To generate a modified macrodipole, we envisioned the addition of another amide as functional group to construct the macrocyclic peptides. This led us to consider the triazole modified cis-β-furanoid dipeptide isostere as an amino acid and analyse its conformation in larger macrocyclic peptides (Fig. 1).

Fig. 1 Incorporation of α- and β-amino acids for the construction of triazole modified macrocyclic peptides 1 and 2.

Replacement of amide by urea while retaining the (1,4)-linked triazole has proved to be an important tool to create greater divergence in macrodipole in pseudo-cyclo-β-peptides. We therefore envisioned that increase in the number of such functional groups in macrocyclic peptides may generate a modified macrodipole leading to a predictable self-assembly pattern. Finally, considering the stability of one particular rotameric conformation of 1,4-linked triazole sugar amino acids (Fig. 2),^6b we decided to synthesize cyclic pseudo peptides with incorporation of α-D-amino acids as well as β-amino acids.⁸ Our study highlights the successful synthesis of novel macrocyclic peptides incorporating D-α-amino acid (1) or β-amino acid (2). While 1 conformationally belongs to the novel class of pseudo cyclic peptides, 2 forms only a conformationally homogeneous cyclic peptide. It therefore appears likely that the macrodipole of self-assembling peptide nanotube can be improved (Scheme 1) by the expansion of peptidomimetic macrocycle (3) to backbone modified macrocyclic peptide 1.^3b

Fig. 2 Possible rotameric conformations of 1,4-linked triazole sugar amino acids.

Scheme 1 Synthetic modification of triazole/amide based peptidomimetic macrocycle 3 by the introduction of amino acids in macrocyclic peptide 1, with macrodipole modification as brought out in 3′ and 1′.

The basic strategy for the solution phase synthesis of the macrocyclic peptide is a Cu(I) catalyzed azide/alkyne cycloaddition⁹ to prepare the 1,2,3-triazole tri-β-peptide isostere (Scheme 2). Cleavage of one acetonide ring of diacetone glucose with AcOH : H₂O (3 : 1) followed by oxidative cleavage of the diol derivative by treatment with sodium metaperiodate furnished the aldehyde 4. Subsequent Ohira–Bestmann reaction yielded the sugar amine derivative 5. Compound 6 could be readily synthesized from a sugar amino alkyne¹⁰ by coupling with Boc protected D-alanine using a standard protocol (employing EDC·HCl, HOBt and DIEA). Compound 7, obtained from diacetone glucose following a well established protocol,¹¹ was then coupled with 6 to yield the intermediate 8.

Scheme 2 Synthesis of basic intermediate 8.

Compound 8 was converted (Scheme 3) to two different intermediates: (i) the amino acid derivative 9 by H₂/Pd–C treatment, and (ii) the amino ester 10 by Boc deprotection with TFA : DCM (1 : 5). Coupling the two intermediates by standard protocol (using EDC·HCl, HOBt and DIEA) furnished the compound 11, which was hydrogenolyzed by H₂/Pd–C to obtain the Boc protected amino acid. Activation of the acid by pentafluorophenol, Boc deprotection with TFA : DCM (1 : 5), and cyclization with DIEA in CH₃CN then gave the targetted triazole/amide macrocyclic compound 1 which was purified by preparative HPLC. The other compound (2) was prepared by employing almost the same methodology (Scheme 4) but starting with a β-amino acid.

Scheme 3 Solution phase synthesis of macrocyclic peptide 1.

Scheme 4 Solution phase synthesis of macrocyclic peptide 2.

¹H NMR spectrum of the D-α-alanine based macrocyclic peptide 1 recorded in the polar solvent CD₃CN and nonpolar solvent such as CDCl₃ : CCl₄ (2 : 3), showed sharp and well-resolved signals, which testified to a consistent and predominant conformation with high degree of C₂ symmetry. The coupling constants of amide proton (NH) with adjacent proton of sugar and also alanine were observed to be large (J_NH,SβH = J_NH,αH = 7.2 Hz), suggesting an antiperiplanar arrangement with flat ring shape required for intermolecular backbone to backbone H-bonding. However, the intensity of ROE cross peaks of the signals of the triazole ring proton with C(β) proton of sugar S1 was strong, when that with signal of C(α) proton of sugar S2 was weak. This indicated a pseudo trans conformation between amine and acid parts of the cis-β-furanoid sugar amino acid.

Molecular modelling was carried out to find the energy minimized conformation of the cyclic peptide with inclusion of distance restraints and dihedral constraints derived via ¹H as well as multidimensional NMR spectroscopy studies. It revealed that the cyclic peptide structure tends to minimize intramolecular side chain-side chain and side chain-backbone interactions by adopting a flat ring-shaped conformation (Fig. 3). It also suggested the distinct conformations of the two faces: NH and C=O groups of the furanoid sugar triazole amino acid (FSTAA face) and NH and C=O groups of α-D-amino acid of the hybrid cyclic peptide backbone (α-D-amino acid face). The amide and triazole units (α-D-amino acid face) remained perpendicular to the mean plane of the peptide ring, whereas the other amide (FSTAA) remained in the same plane as the peptide backbone. The NH and C=O groups of the α-amino acid residues lie in the same direction with (1,4)-linked triazole. As a result, the conformation is likely to impart higher macrodipole to the synthesised cyclic peptide 1 compared to the (α,ε) cyclic peptides.^3b

Fig. 3 Possible energy minimized structures of (a) pseudo cyclic peptide 1 and (b) cyclic peptide 2 by molecular modelling (using CHARMm force field on Discovery Studio 4.0).

In the FT-IR spectrum, presence of amide I and amide II bands at 1670 and 1531 cm⁻¹, respectively, suggested the expected flatness of the peptide ring. Location of the amide A NH band at 3325 cm⁻¹ indicated the formation of a β-sheet-like structure as previously reported for cyclic peptides as well as peptidomimetic macrocycles.^6,11

The macrocyclic peptide 1 was dissolved in 2 : 3 CDCl₃–CCl₄. The solution turned cloudy and this was used for analysis by NMR and FT-IR to confirm self-assembly. SEM and AFM morphologies were then studied to observe nanorod formation.¹¹ The images show rod-like assemblies, the surfaces of which appear smooth (Fig. 4). The diameters are in the range of 100–500 nm and lengths over several microns.^6d

Fig. 4 SEM images (a) and (b) of rod like assemblies of 1; the AFM images (c) and (d) are in (2 : 3) CDCl₃ : CCl₄.

Though the actual mechanism of nanorod formation is not known, it may be similar to the “hierarchical process” proposed by Dory and co-workers involving an assembly of individual PNTs which over several generations forms “needle-shaped crystals”. The force associated with this process is speculated to originate from non-covalent inter-tube interactions (hydrophobic, van der Waals) and electrostatic interactions involving dipoles.^6e,f

To characterise the self assembly pattern of 1, the ¹H NMR spectrum was recorded in a nonpolar solvent such as CDCl₃ : CCl₄ (2 : 3). In contrast to that in a polar solvent, it showed broadening of backbone proton signals which is attributed to intermolecular H-bond mediated aggregation exchanging on NMR time scale, giving rising to a supramolecular species.^3b,6 To determine the relative orientation of peptide backbone units in nanorod structure, the ROESY spectrum was analysed carefully. Definitive evidence in favour of parallel stacking came from the observed cross peaks between S2-CHα and S2-CHδ peaks of the furanoid sugar moiety (Fig. 5 and 6).

Fig. 5 Typical side view of intermolecular H-bonding in 1 derived by molecular modelling. The right part shows ROESY correlations observed in (2 : 3) CDCl₃ : CCl₄.

Fig. 6 Selected region of the ROESY spectrum of 1 showing parallel-homostacking interaction by cross peaks between S2Hα–S2Hδ in (2 : 3) CDCl₃ : CCl₄ (600 MHz, 298 K).

¹H NMR spectra of macrocycle 2 in both nonpolar (CDCl₃) and polar (CD₃CN) solvents were also well-defined and predictive of a C₂ symmetric nature. The large coupling constant J_NH,SβH = 8.4 Hz was like that in pseudo cyclo peptide 1 and typical of the all trans backbone conformation required for the flatness of the cyclic peptide. The coupling constants of the other amide NH (β-alanine NH), 3.6 and 10.2 Hz for the two adjacent diastereotopic protons, indicated a nearly antiperiplanar arrangement of the peptide backbone. The ROESY cross-peaks between β-Ala NH and α-CH of the sugar moiety furnished evidence for the opposite orientation of two amide bonds in peptide 2 (Fig. 3).

Molecular modelling study revealed that the amide NH and carbonyl group occupy the same face while β-Ala NH and C=O remain on the other (see ESI†). But unlike macrocyclic peptide 1, the amide groups are not oriented perpendicular to the backbone of the ring which was substantiated by FT-IR spectroscopy showing amide I and amide II bands at 1671and 1532 cm⁻¹, respectively. This ruled out the flatness of the peptide ring. In addition, the position of the amide A band (3346 cm⁻¹) excluded the possibility of self assembly.¹² Solution phase ROESY of compound 2 also did not show any cross-peak in support of self-assembly in polar as well as nonpolar solvents,⁶ unlike macrocyclic peptide 1.

AFM and SEM studies were not considered for 2 as the solutions did not turn cloudy and NMR and FT-IR did not support self-assembly.

In conclusion, we have designed and synthesized two types of novel heterocyclic backbone modified macrocyclic peptides by incorporation of an α-amino acid/β-amino acid with cis-β-furanoid (1,4)-linked triazole amino acid. The macrocyclic peptide 1 featuring α-amino acid is able to maintain pseudo cyclo-peptide conformation with predictable self-assembly pattern, while 2 based on β-amino acid forms only a conformationally homogeneous cyclic peptide that did not undergo self-assembly. Peptide 1 can undergo parallel homo-stacking via amide NH and amide carbonyl oxygen H-bonding like cyclo-β-peptides and the unidirectional assembly pattern allows such macrocycles to possess larger dipole moments. This type of macrocycles may serve as model systems for different classes of artificial ion channels.

Acknowledgements

Authors thank CSIR for research fellowship (to G. K.), CSIR-HOPE and ORIGIN Projects (Govt of India) for financial support, Dr Chitra Dutta of Structural Biology and Bio-Informatics Division of this institute for permitting use of Discovery Studio for molecular modelling and Dr Krishnananda Chattopadhyay of the same Division for helpful suggestions.

References

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra11850f

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