Residue dependent hydrogen-bonding preferences in orthanilic acid-based short peptide β-turn motifs

Abstract

This communication describes the competition between native β-turn (C10) and 2-aminobenzenesulfonic acid (^SAnt)(orthanilic acid)-based pseudo β-turn (C11) in their hybrid peptides. Solid-state crystal structure and solution-state NMR studies revealed that C10 and C11 can be simultaneously observed under appropriate conditions. The variable temperature NMR coefficient data suggest that the isolated C11/C14 hydrogen bond is weaker in comparison with the consecutive C10 and C11 turns.

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Peptides occur throughout nature and play wide-ranging roles in many processes, though, their utility as drugs is limited, primarily because of their intrinsic conformational flexibility.¹ Indeed, a wide spectrum of research has attracted much attention to peptides involving modulation of their properties through peptide modification and attaining proteolytic stability by conformational restriction.² Modifying the peptide backbone by isosteric peptide bond modifications, N/C-alkylations, short-range cyclisations, the use of synthetic amino acids, conformational modulation by intramolecular interactions, amide to sulfonamide alterations, etc. are some of the approaches by which the rigidification of peptide backbone can be accomplished.³ Several research groups rely on non-peptidic substances for drug discovery, which has led to the development of the field of peptidomimetics.⁴ Meanwhile, the study of non-natural oligomers displaying discrete folding propensities – akin to biopolymers – has paved the way for the development of foldamers.⁵

Pro–Aib (proline and 2-aminoisobutyric acid) often perturbs the normal hydrogen-bonding patterns found in helices owing to their dihedral constraints, giving rise to β-bend structures featuring C10 hydrogen bonding in their sequential hybrid peptides.^{6 L}Pro–^LPro and ^DPro–^LPro induce a helical architecture when placed at the N-terminus of peptide sequences featuring C10 hydrogen bonding.^{7 L}Pro–Gly is another motif featuring the C10 hydrogen bond.^7b However, orthanilic acid (2-aminobenzenesulfonic acid, ^SAnt), which has a very high propensity to induce a β-turn-like structure, known to display robust 11-membered intramolecular H-bonding interactions in the sequences ^LPro–^SAnt/Gly–^SAnt due to the sulfonamide torsional preference.^8a–d More recently, we reported that ^SAnt–^LPro at the C-terminus of β-turn motifs (^DPro–Aib, ^D/LPro–^LPro, Aib–^LPro and ^LPro–Gly) destabilize the native β-turn in those pseudo-peptides.^8e

In order to understand better the role of conformational restriction on the hydrogen-bonding pattern in native β-turn motifs featuring 10-membered hydrogen bonds, ^SAnt was incorporated into the peptide sequences Aib–^LPro–^SAnt 1, Gly–^LPro–^SAnt 2, ^LPro–Aib–^SAnt 3, ^LPro–Gly–^SAnt 4, ^LPro–^LPro–^SAnt 5, ^DPro–^LPro–^SAnt 6 and ^LtLeu–^LPro–^SAnt 7/8. The syntheses of these peptides were carried out using standard solution-phase coupling procedures (ESI,† pages S3–S10). Extensive conformational investigations of the peptides were carried out using solution-state NMR studies and crystal structure/NOE-based MD simulated studies (ESI,† page S29–S97).

The crystal structure of the peptide Piv–Aib–^LPro–^SAnt–NH^IBu 1 displayed the co-existence of two β-turns (i ← i + 3) – i.e. C10 as well as C11 hydrogen bonding running in reverse direction (Fig. 1). This clearly suggests that both the turn motifs hardly interfere with each other and run mutually to stabilize the turn architecture. Here, the direction of both hydrogen bonds remained the same and each carbonyl satisfied an NH for hydrogen bonding in a sequential manner. It was also noted that the ^SAntNH was not involved in C6 intra-residual hydrogen bonding and seldom affected the conformation features of the nearby Aib–Pro reverse turn structure. Extensive solution-state NMR studies of this peptide support a similar conformation in the solution state (ESI,† pages S29–S33). The NOE observed between the NH of ^SAnt and the methyl groups of Aib residue confirms the presence of a C10 hydrogen bond between the NH of ^SAnt and the carbonyl of the N-terminal pivaloyl group (ESI,† page S33). Similarly, the NOE observed between the NH of isobutylsulphonamide and the NH of ^SAnt confirms the presence of a C11 hydrogen bond between the isobutylsulphonamide NH and the carbonyl oxygen of the Aib residue (ESI,† page S33). Furthermore, the NMR temperature coefficients for the NH of ^SAnt (involved in the C10 hydrogen bond) and the NH of isobutylsulphonamide (involved in the C11 hydrogen bond) were −0.18 and −0.36 ppb K⁻¹, respectively (ESI,† page S65). The observed negligible NMR temperature coefficients suggest that these C10 and C11 hydrogen bonds are robust.

Fig. 1 Crystal structure (grey) of 1 NOE based MD simulated model (light blue) (ESI,† pages S93–S96) of 3 and 4 with their chemical structures displaying C10 and C11 hydrogen bonds.

Intense efforts to crystallize the peptides 3 Piv–^LPro–Aib–^SAnt–NH^IBu and 4 Piv–^LPro–Gly–^SAnt–NH^IBu failed to generate good quality crystals for the X-ray diffraction studies. Hence, we relied on solution-state NMR studies for understanding the conformations of these peptides (ESI,† pages S39–S48). In peptide 3, the NOE observed between the NH of ^SAnt and the N-terminal pivaloyl methyl groups confirms the presence of a C10 hydrogen bond, and furthermore, the NOE observed between the NH of isobutylsulphonamide and the α-hydrogen of the L-proline confirms the presence of the C11 hydrogen bond (ESI,† page S43). Similarly, in peptide 4, the NOE observed between the NH of ^SAnt and the N-terminal pivaloyl methyl groups confirms the presence of the C10 hydrogen bond, and furthermore, the NOE observed between the NH of isobutylsuphonamide and the α-hydrogens of glycine confirms the presence of the C11 hydrogen bond (ESI,† page S48). The NMR temperature coefficients in peptide 3 for the NH of ^SAnt (involved in the C10 hydrogen bond) and the NH of isobutylsulphonamide (involved in the C11 hydrogen bond) were −0.18 and −1.09 ppb K⁻¹, respectively (ESI,† page S69). Similarly, the NMR temperature coefficients in peptide 4 for the NH of isobutylsulphonamide (involved in the C11 hydrogen bond) is −1.27 ppb K⁻¹ and there was no shift in the NH of ^SAnt (ESI,† page S69). The observed negligible NMR temperature coefficients in peptides 3 and 4 suggest that these C10 and C11 hydrogen bonds are robust. The distance constraints utilized for building an NOE-based model for these peptides are listed in ESI,† pages S94–S96. The NOE-based model studies of 3 and 4 showed evidence of the simultaneous existence of two β-turns (i ← i + 3) i.e. C10 and C11 hydrogen bonds in a reverse direction, similar to peptide 1 (Fig. 1) (ESI,† pages S93–S96).

Replacement of the Aib residue in peptide 1 by ^LPro/^Lt-Leu, as in peptides 5 and 7/8, respectively, resulted in the disappearance of the C10 hydrogen bond, while, C11 hydrogen bond was intact. The destabilization of the 10-membered hydrogen bonding in 5 and 7/8 was presumably due to the dihedral constraints of the ^LPro–^LPro residues and the bulky nature of the tertiary butyl side chain in 5 and 7, respectively (Fig. 2). A similar conformation of 5 and 7 existing in the solution state was evident from the NMR studies (ESI,† pages S49–S53, S59–S63). The NOE observed between the NH of isobutylsulphonamide and the NH of ^SAnt residue confirmed the presence of the C11 hydrogen bond in peptides 5 and 7, respectively (ESI,† pages S53 and S63). For peptides 5 and 7, the observed NMR temperature coefficients for the NH of isobutylsulphonamide involved in the C11 hydrogen bonding were −2.727 and −4.55 ppb K⁻¹, respectively (ESI,† pages S73 and S77). The NMR temperature coefficient values of peptides 5 and 7 suggest that the C11 hydrogen bond is weaker in comparison with the same bond in peptides 1, 3 and 4 (these have both C10 and C11). The crystal structure of 8 has two molecules with slightly different orientations in the unit cell – though both molecules displayed C11 and C6 hydrogen bonds. Furthermore, in peptides 5, 7 and 8, the ^SAnt residue displayed intra-residual C6 hydrogen bonding suggesting that the co-existence of C10 and C11 turns can be possible in the absence of conformational preferences offered by C6 hydrogen bonding in ^SAnt. This observation was in agreement with the secondary structure of 2 and 6, which displayed C6 and C14 hydrogen bonded networks (Fig. 3). Furthermore, in peptide 7, an additional intra-residual C5 hydrogen bond was observed in the L-tert-Leu residue with the NMR temperature coefficient of −0.07 ppb K⁻¹ (ESI,† page S77).

Fig. 2 NOE-based MD simulated model (light blue) (ESI,† pages S93, S96–S97) of 5 and crystal structures (grey) of 7 and 8, with their chemical structures displaying C6, C11 hydrogen bonds. Additional intra-residual C5 hydrogen bond was observed in tert-Leu residue in peptide 7.

Fig. 3 Crystal structures (grey) of 2 and 6 with their chemical structures displaying C6 and C14 hydrogen bonds.

Replacement of the conformationally constrained Aib residue at the N-terminus in 1 by the flexible amino acid Gly in peptide 2 resulted in a drastic shift of the conformational features from β-turns (C10 and C11) to an extended α-turn-like C14 hydrogen bond (i ← i + 4) along with a C6 intra-residual hydrogen bond in ^SAnt (Fig. 1 and 3). In another instance, chirality alteration of the N-terminus from L-proline (5) to D-proline (6) resulted in a shift of the β-turn (C11) (i ← i + 3) to an extended α-turn-like C14 hydrogen bond (i ← i + 4) by retaining the C6 intra-residual hydrogen bond in ^SAnt (Fig. 2 and 3). Similar conformation was observed in the solution state as evident from the NMR studies (ESI,† pages S34–S38 and S54–S58). In peptides 2 and 6, the NOE observed between the NH of isobutylsulphonamide and the methyl groups of the N-terminal pivaloyl group confirmed the presence of a C14 hydrogen bond between the NH of isobutylsulphonamide and the carbonyl oxygen of the N-terminal pivaloyl group (ESI,† pages S38 and S58). For peptides 2 and 6, the NMR temperature coefficients of isobutylsulphonamide NH involved in the C14 hydrogen bond were −1.27 and −4.91 ppb K⁻¹, respectively.

Table 1 shows torsional parameters observed in the crystal structure of compounds 1, 2, 6, 7 and 8 (ESI,† pages S78–S92). The (ϕ, ψ) values of the Aib residue were highly restricted in peptide 1 and were observed in the range of Ramachandran angles⁹ (α-helix, ϕ ≈ −57° and ψ ≈ −47°; 3₁₀ helix, ϕ ≈ −50° and ψ ≈ −30°).¹⁰ Furthermore, the (ϕ, ψ) values for L-proline were in the most observed values i.e. ϕ = −60° (±20°), ψ = −30° (±20°) and ϕ = −60° (±20°), ψ = 120° (±30°), from the distribution of 4996 proline residues in high resolution protein crystal structures deposited in the Protein Data Bank,¹¹ except ψ = −0.4° in peptide 1, which was slightly different and featured two β turns (C10 and C11). Also, the ϕ = −87° of ^SAnt in the peptide 1 was exceptional in the series, compared to the other peptides with ϕ = 140° (±30°) and ϕ = −160° (±30°) featuring two β turns (C10 and C11). The hydrogen-bonding distances [d(H⋯A)] for C10 and C11 in peptide 1 were 1.89 Å and 2.07 Å, indicating the robustness of the fold. The sulfonamide torsion ω was found to be ∼90°, as in the majority of known sulfonamide crystal structures, indicating the high folding preference of sulfonamide torsion.¹²

Table 1 Backbone torsional angles observed in the crystal structures of 1, 2, 6, 7 and 8

Structures	Torsion^a (°)								Type of inter-residual H bonding	Torsion angle C=O⋯H–N (°)	H-Bonding distance NH⋯O (Å)
	Xaa (Aib, Gly, ^DPro, ^LtLeu)		Yaa (^LPro)		^SAnt
	ϕ	ψ	ϕ	ψ	ϕ	θ	ψ	ω
a Note: for further details see: ESI,† pages S10–S12 and S78–S92.^14
1	−47.8(6)	−47.0(6)	−78.0(5)	−0.4(6)	−87.0(6)	−1.6(6)	−58.7(4)	−59.6(4)	C10	161.4	1.89
									C11	179.4	2.07
2	84.4(4)	−177.9(3)	−81.4(3)	−20.8(4)	−165.3(3)	4.2(4)	67.3(3)	72.6(2)	C14	−151.4	2.21
6	66.9(4)	−161.2(2)	−85.2(3)	−14.9(4)	163.4(3)	2.9(4)	81.6(3)	76.5(3)	C14	−91.6	2.03
7	−121.0(3)	138.7(3)	−64.6(3)	140.9(3)	130.7(3)	−3.7(4)	−68.3(3)	−71.8(3)	C11	141.2	2.21
8	−128.4(3)	147.6(3)	−63.3(4)	150.7(3)	120.0(3)	−7.3(4)	−54.7(3)	−52.4(3)	C11	39.3	2.46
	−110.1(3)	132.0(3)	−58.7(4)	140.7(3)	123.2(4)	−4.8(5)	−61.5(3)	−64.4(3)	C11	80.2	2.35

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In conclusion, we have described the competition between the β-turns, i.e. C10 and C11 hydrogen bonding with overlapping turn residues. Crystal structure and solution-state NMR studies of peptide 1 clearly revealed the co-existence of C10 and C11, presumably due to the backbone torsional preferences of the Aib–Pro–^SAnt sequence. Peptides 3 and 4 displayed conformations similar to 1, owing to the presence of highly stable Pro–Aib and Pro–Gly (C10 turn) and a comparatively flexible ^SAnt residue with a pseudo β-turn (C11). Furthermore, peptides 1, 3 and 4 did not display intra-residual C6 hydrogen bonding in ^SAnt. Conformational restriction offered by the intra-residual C6 hydrogen bond of ^SAnt played a pivotal role in attaining the three-dimensional conformations of 2 and 5–8. The peptides 5, 7 and 8 revealed the presence of C11 hydrogen bonds (absence of C10), while 2 and 6 showed the presence of a C14 hydrogen bond (absence of C10 as well as C11). These findings suggest that under appropriate conditions, it is possible to stabilize both the β-turn and pseudo β-turn from non-proteinogenic residues such as ^SAnt with overlapping turn residues, and this may be incorporated into bioactive peptides similar to other reverse turns.¹³

Acknowledgements

GSJ, KNV, ASK, ES, and DRS thank CSIR (New Delhi) for their research fellowships. GJS thanks CSIR BIODISCOVERY project (BSC-0120) for financial support.

Notes and references

1. (a) J. M. Mason, Future Med. Chem., 2010, 2, 1813; (b) J. Vagner, H. Qu and V. J. Hruby, Curr. Opin. Chem. Biol., 2008, 12, 292.
2. (a) J. S. Blakeney, R. C. Reid, G. T. Le and D. P. Fairlie, Chem. Rev., 2007, 107, 2960; (b) M. J. M. Niesen, S. Bhattacharya and N. Vaidehi, J. Am. Chem. Soc., 2011, 133, 13197; (c) I. E. Andersson, T. Batsalova, S. Haag, B. Dzhambazov, R. Holmdahl, J. Kihlberg and A. Linusson, J. Am. Chem. Soc., 2011, 133, 14368.
3. (a) C. Gennari, B. Salom, D. Potenza and A. Williams, Angew. Chem., Int. Ed., 1994, 33, 2067; (b) C. Caumes, O. Roy, S. Faure and C. Taillefumier, J. Am. Chem. Soc., 2012, 134, 9553; (c) C. Tomasini, G. Angelici and N. Castellucci, Eur. J. Org. Chem., 2011, 3648; (d) J. R. Stringer, J. A. Crapster, I. A. Guzei and H. E. Blackwell, J. Org. Chem., 2010, 75, 6068; (e) R. M. J. Liskamp, D. T. S. Rijkers, J. A. W. Kruijtzer and J. Kemmink, ChemBioChem, 2011, 12, 1626; (f) S. Turcotte, S. H. B. Gervais and W. D. Lubell, Org. Lett., 2012, 14, 1318; (g) T. D. W. Claridge, D. D. Long, C. M. Baker, B. Odell, G. H. Grant, A. A. Edwards, G. E. Tranter, G. W. J. Fleet and M. D. Smith, J. Org. Chem., 2005, 70, 2082.
4. (a) A. Brust, C. A. Wang, N. L. Daly, J. Kennerly, M. Sadeghi, M. J. Christie, R. J. Lewis, M. Mobli and P. F. Alewood, Angew. Chem., Int. Ed., 2013, 52, 1; (b) M. A. Scorciapino and A. C. Rinaldi, Front. Immunol., 2012, 3, 1; (c) E. Ko, J. Liu and K. Burgess, Chem. Soc. Rev., 2011, 40, 4411; (d) P. G. Vasudev, S. Chatterjee, N. Shamala and P. Balaram, Chem. Rev., 2011, 111, 657; (e) C. Andre, B. Legrand, C. Deng, C. Didierjean, G. Pickaert, J. Martinez, M. C. Averlant-Petit, M. Amblard and M. Calmes, Org. Lett., 2012, 14, 960; (f) B. Song, M. G. Bomar, P. Kibler, K. Kodukula and A. K. Galande, Org. Lett., 2012, 14, 732; (g) S. Kushal, B. B. Lao, L. K. Henchey, R. Dubey, H. Mesallati, N. J. Traaseth, B. Z. Olenyuk and P. S. Arora, Proc. Natl. Acad. Sci. U. S. A., 2013, 110, 15602.
5. (a) V. Azzarito, K. Long, N. S. Murphy and A. J. Wilson, Nat. Chem., 2013, 5, 161; (b) I. Saraogi and A. D. Hamilton, Chem. Soc. Rev., 2009, 38, 1726; (c) D. W. Zhang, X. Zhao, J. L. Hou and Z. T. Li, Chem. Rev., 2012, 112, 5271; (d) G. Guichard and I. Huc, Chem. Commun., 2011, 47, 5933; (e) P. Prabhakaran, S. S. Kale, V. G. Puranik, P. R. Rajamohanan, O. Chetina, J. A. K. Howard, H.-J. Hofmann and G. J. Sanjayan, J. Am. Chem. Soc., 2008, 130, 17743; (f) R. V. Nair, S. Kheria, S. Rayavarapu, A. S. Kotmale, B. Jagadeesh, R. G. Gonnade, V. G. Puranik, P. R. Rajamohanan and G. J. Sanjayan, J. Am. Chem. Soc., 2013, 135, 11477; (g) A. Roy, P. Prabhakaran, P. K. Baruah and G. J. Sanjayan, Chem. Commun., 2011, 47, 11593; (h) G. Priya, A. S. Kotmale, R. L. Gawade, D. Mishra, S. Pal, V. G. Puranik, P. R. Rajamohanan and G. J. Sanjayan, Chem. Commun., 2012, 48, 8922; (i) T. A. Martinek and F. Fülöp, Chem. Soc. Rev., 2012, 41, 687.
6. B. Di Blasio, V. Pavone, M. Saviano, A. Lombardi, F. Nastri, C. Pedone, E. Benedetti, M. Crisma, M. Anzolin and C. Toniolo, J. Am. Chem. Soc., 1992, 114, 6273.
7. (a) R. Rai, S. Aravinda, K. Kanagarajadurai, S. Raghothama, N. Shamala and P. Balaram, J. Am. Chem. Soc., 2006, 128, 7916; (b) E. G. Hutchinson and J. M. Thornton, Protein Sci., 1994, 3, 2207.
8. (a) K. N. Vijayadas, H. C. Davis, A. S. Kotmale, R. L. Gawade, V. G. Puranik, P. R. Rajamohanan and G. J. Sanjayan, Chem. Commun., 2012, 48, 9747; (b) S. S. Kale, G. Priya, A. S. Kotmale, R. L. Gawade, V. G. Puranik, P. R. Rajamohanan and G. J. Sanjayan, Chem. Commun., 2013, 49, 2222; (c) V. V. E. Ramesh, S. S. Kale, A. S. Kotmale, R. L. Gawade, V. G. Puranik, P. R. Rajamohanan and G. J. Sanjayan, Org. Lett., 2013, 15, 1504; (d) S. S. Kale, S. M. Kunjir, R. L. Gawade, V. G. Puranik, P. R. Rajamohanan and G. J. Sanjayan, Chem. Commun., 2013, 49, 2222; (e) T. S. Ingole, K. N. Vijayadas, K. N. Chaitanya, A. S. Kotmale, R. L. Gawade, R. G. Gonnade, P. R. Rajamohanan and G. J. Sanjayan, Eur. J. Org. Chem., 2016, 1380.
9. (a) G. N. Ramachandran, C. Ramakrishnan and V. Sasisekharan, J. Mol. Biol., 1963, 7, 95; (b) G. N. Ramachandran and V. Sasisekharan, Adv. Protein Chem., 1968, 23, 283.
10. S. Mammi, M. Rainaldi, M. Bellanda, E. Schievano, E. Peggion, Q. B. Broxterman, F. Formaggio, M. Crisma and C. Toniolo, J. Am. Chem. Soc., 2000, 122, 11735.
11. F. C. Bernstein, T. F. Koetzle, G. J. Williams, E. E. Meyer Jr, M. D. Brice, J. R. Rodgers, O. Kennard, T. Shimanouchi and M. Tasumi, J. Mol. Biol., 1977, 112, 535.
12. A. Parkin, A. Collins, C. J. Gilmore and C. C. Wilson, Acta Crystallogr., Sect. B: Struct. Sci., 2008, 64, 66.
13. G. S. Jedhe, A. S. Kotmale, P. R. Rajamohanan, S. Pasha and G. J. Sanjayan, Chem. Commun., 2016, 52, 1645.
14. CCDC 1440080–1440084 (1, 2, 6–8).†.

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Footnote

† Electronic supplementary information (ESI) available: General experimental procedures, ¹H, ¹³C, DEPT-135, COSY, TOCSY, HMBC, HSQC, NOESY NMR spectra, HRMS spectra crystal data of all new compounds. CCDC 1440080–1440084. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra05684a

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