Probing the folding induction ability of orthanilic acid in peptides: some observations

Abstract

This paper describes the ability of orthanilic acid (2-aminobenzenesulfonic acid, ^SAnt) to promote folding when introduced in a peptide sequence. Three peptide sequences, containing orthanilic acid (^SAnt) with a sulfonamide moiety in the turn segment, have been synthesized in the solution phase using suitable coupling agents, and their structural aspects investigated using NMR and X-ray crystallographic studies. Solid- and solution-state conformational analyses reveal that the peptide sequences containing orthanilic acid in their backbone exist in a folded conformation featuring long-range 15-membered ring H-bonding.

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Introduction

Unnatural amino acids are vital for understanding and mimicking bio-constructs. In the recent past, chemists have taken advantage of using unnatural amino acids to develop peptidomimetic-based leads for drug discovery.¹ This is primarily because of the proteolytic stability of the unnatural amino acids as compared to their natural counterparts, which makes them unsuitable to be used in therapeutics. Moreover, the incorporation of unnatural amino acids in a peptide sequence offers the possibility of generating diverse secondary structures. Over the years, various research groups have developed and utilized unnatural amino acids to generate numerous peptidomimetic and foldamer molecules with interesting secondary structural preferences (Fig. 1).^2–4

Fig. 1 Some of the important unnatural amino acids which have frequently been used for generating various peptidomimetic molecules.

Of the amino acids shown in Fig. 1, orthanilic acid deserves a special mention, because ^SAnt can introduce a sulfonamide bond into a poly-peptide chain. A sulfonamide bond possesses higher proteolytic stability, polar character, and a tetrahedral sulfur atom mimicking the intermediate formed during proteolysis. Sulfonamides therefore make up an important class of drugs, with many pharmacological agents showing antibacterial, antitumor, anticarbonic anhydrase, diuretic, hypoglycemic, antithyroid, or protease inhibitory activity,⁵ and many more.⁶ Although similar, sulfonamides and carboxamides have substantial conformational disparity because of the presence of an additional H-bond acceptor. Moreover, the rotation barrier about the S–N bond is much lower as compared to the C–N bond, which makes sulfonamides more flexible, leading them to remain in a twisted conformation which may induce folding.⁷ Furthermore, the differences in the dihedral angles of amides and sulfonamides are reason enough for them to be used as a peptide bond replacement to generate intriguing structures. Sulfonamides, as a result, have long been used as an amide bond surrogate to develop various synthetic peptides.⁸

Result and disscusion

We have previously demonstrated the geometrical and H-bonding similarity between sulfonamide and carboxamide by inserting orthanilic acid (^SAnt) in place of anthranilic acid (Ant) in the Ant–Pro (anthranilic acid–proline) turn segment which showed the existence of strong 9-membered ring H-bonding.⁹ The results obtained were quite intriguing given the fact that these groups have relatively different H-bonding and geometrical preferences, which is well established.¹⁰ However, the literature precedents and the geometric disparity of sulfonamides as compared to carboxamides are too significant to be content with a single result. Consequently, to explore further in the direction of developing novel peptidomimetics and understanding their conformational preferences, we designed a range of short chain oligopeptides featuring ^SAnt–Pro as the potential turn inducer (Fig. 2).

Fig. 2 Design strategy to synthesize oligomer sequences 9, 12 and 15 using orthanilic acid as a connecting entity to facilitate folding.

Synthesis

The synthesis of the foldamer sequence bearing orthanilic acid (^sAnt) started with dimer 1 (Scheme 1), which on catalytic hydrogenation furnished amine 2 in quantitative yield. Coupling of amine 2 with 2-bromo-2-methylpropanoyl bromide gave 3 in 90% yield, which was subjected to nucleophilic substitution to furnish azide 4 in very good yield. The trimer azide 4 was then reduced in the presence of H₂, Pd–C and coupled with Boc–Leu–OH in the presence of EDC·HCl as the coupling agent to obtain the tetramer 6a in 85% yield. Ester hydrolysis of 6a was followed by coupling with the free amine of dipeptide 7,¹¹ furnishing hexamer 8 in 75% yield. Hexamer 8 on treatment with saturated methanolic MeNH₂ produced the methyl amide analog 9 in 80% yield. The results obtained during the conformational analyses (vide infra) of oligomer 9 suggested that the Leu₁ residue at the C-terminus had frayed, presumably owing to the inherent flexible nature of leucine. This prompted us to replace the flexible Leu residue at the N-terminus with the conformationally rigid Aib and Pro, developing two more sequences.

Scheme 1 Reagents and conditions: (i) H₂, Pd–C, MeOH, 12 h; (ii) 2-bromo-2-methylpropanoyl bromide, Et₃N, DCM, 12 h, 90%; (iii) NaN₃, DMF, 75 °C, 12 h, 81%; (iv) H₂, Pd–C, 60 psi, 10 h; (v) Boc–Leu–OH, EDC, HOBt, DCM, 10 h, 85%; (vi) LiOH, MeOH, H₂O, 4 h; (vii) EDC, HOBt, DCM, 10 h, 75%; (viii) sat. methanolic MeNH₂, 8 h, 80%.

To prepare the sequence with α-aminoisobutyric acid (Aib) at the N-terminus, we started with the trimer amine 5 (Scheme 2) which was coupled with Boc–Aib–OH, in the presence of EDC·HCl as the coupling agent to obtain the tetramer 10a in 85% yield. Ester hydrolysis of 10a was followed by coupling the acid with the free amine of dipeptide 7, producing hexamer 11 in 82% yield. Hexapeptide 11, containing the constrained amino acid Aib at the N-terminus, upon treatment with sat. methanolic MeNH₂ furnished the methyl amide analog 12 in 82% yield.

Scheme 2 Reagents and conditions: (i) Boc–Aib–OH, EDC, HOBt, DCM, 10 h, 85%; (ii) LiOH, MeOH, H₂O, 4 h; (iii) 7, EDC, HOBt, DCM, 10 h, 82%; (iv) sat. methanolic MeNH₂, 8 h, 82%; (v) Piv–Pro–OH, EDC, HOBt, DCM, 10 h, 86%; (vi) LiOH, MeOH, H₂O, 4 h; (vii) 7, EDC, HOBt, DCM, 10 h, 73%; (viii) sat. methanolic MeNH₂, 8 h, 86%.

In order to access another sequence and obtain 15 bearing proline at the N-terminus, free amine 5 (Scheme 2) was first coupled with Piv–Pro–OH, furnishing the tetramer 13a in 86% yield. Free acid 13b, obtained after the basic hydrolysis of tetramer 13a, was coupled with the free amine of dipeptide 7 to get the hexamer 14 in 73% yield, which on reaction with sat. methanolic MeNH₂ resulted in the formation of the methyl amide analog 15 in 86% yield.

Solid-state conformational analysis

Extensive efforts to crystallize the oligomers resulted in the formation of crystals of 9 (Fig. 3).¹² Careful analysis of the crystal data revealed the presence of three inter-residue H-bonding locations, and a 6-membered intra-residue H-bonded ring between NH and S=O of the orthanilic acid moiety [d(C=O⋯H–N) = 1.98 Å, bond angle (N–H⋯O) = 141°]. Of the three different types of inter-residue H-bonding patterns, (i) one was a 10-membered ring (C10 H-bonding) between the C-terminus amide NH and the carbonyl of the proline moiety [d(C=O⋯H–N) = 2.24 Å, bond angle (N–H⋯O) = 130°], (ii) one was a 7-membered ring between S=O of ^SAnt and N–H of Leu₂ [d(C=O⋯H–N) = 2.71 Å, bond angle (N–H⋯O) = 123°], and (iii) one was an unusual, long range (15-membered) H-bonding ring between N–H of Aib₁ and C=O Leu₂ [d(C=O⋯H–N) = 2.02 Å, bond angle (N–H⋯O) = 167°] (Fig. 3). The dihedral angle values shed more light on the observed conformation of the oligomer 9 containing the sulfonamide connecting entity. As evident from the crystal structure, the torsion angles of ^SAnt were: ϕ = 174.68°, θ = 5.01°, ψ = 92.55° and ω = −55.31°. In the case of proline, ϕ and ψ were found to be −107.85° and 19°, respectively. Thus, the solid-state conformational analysis clearly indicates that the torsional constraint of the orthanilic acid (^SAnt) residue is a key factor for the folded conformation seen in oligomer 9.

Fig. 3 Crystal (a) and molecular structure (b) of oligomer 9, and different types of H-bonding observed in the solid-state (c–f).

Conformational investigation in solution-state

We undertook extensive NMR studies to provide insights into the solution-state conformation of the oligomers. Details of the peak assignments with spectra are provided in the ESI.†

All of the oligomers were readily soluble in nonpolar organic solvents (≫100 mM in CDCl₃) at ambient temperature. Signal assignments were made unambiguously using a combination of two-dimensional COSY, TOCSY, HSQC, HMBC and NOESY experiments. Oligomers 9 and 12 exhibited sharp signals for Boc–CH₃ in ¹H NMR, whereas the oligomer 15 with proline at the N-terminus showed multiple signals, suggesting the presence of multiple conformers.

The characteristic nOes supporting the folded conformation of 9 found in the solid-state were the inter-residue coupling between Aib₁–CH₃ (C10H) and NH5 along with Aib₁–CH₃ (C10H) and C33H (ESI, Fig. 6, S39†). Other important nOes were found between Pro–αH (C18H) and ^SAnt (C14H). Dipolar coupling was also observed between Pro–αH (C18H) with NH3 and NH4. However, there were no nOes observed between Boc–CH₃ and the C-terminus methyl (C33H) as anticipated from the single crystal X-ray diffraction analysis.

For the oligomer 12, nOes that supported the folded conformation were between Pro–αH (C16H) and NH3, NH3 and NH4, and C33H and Aib₂–CH3 (C8H). Inter-residue nOes were also observed between NH3 and NH2 (ESI, Fig. 11, S44†).

In the case of oligomer 15, inter-residue dipolar couplings were observed between Pro₂–αH (C17H) and NH2, Pro₂–αH (C17H) and Aib₁–CH₃ (C9H), and C-terminus methyl (C33H) and Aib₁–CH₃ (C9H). Also observed were nOes of Pro₁–αH (C2H) with NH2 and Aib₂–CH₃ (C30H) (ESI, Fig. 16, S49†). Similar nOe patterns of the identical central residues in all the oligomers suggested the prevalence of the folded conformation as anticipated.

To investigate the existence of intramolecular hydrogen-bonding in the oligomers, we also undertook DMSO-d₆ titrations and variable temperature experiments (ESI, S31–S34†). All of the NHs of 9 involved in intramolecular hydrogen bonding showed very little shift in the titration studies [Δδ (NH) < 0.25 ppm]. Only NH5 underwent a relatively larger chemical shift change [Δδ (NH5) < 0.7 ppm] on the incremental addition of DMSO-d₆, suggesting its non-participation in H-bonding. Similar was the case for oligomer 12 where a considerable change in chemical shift was observed for NH5 and NH1 [Δδ (NH) < 0.52 ppm], while the rest of the NHs showed a negligible shift [Δδ (NH) < 0.18 ppm]. Oligomer 15, on the other hand, showed very little change in the chemical shift values for all of the NHs [Δδ (NH) < 0.25 ppm], except for NH3 [Δδ (NH) < 0.49 ppm], implying the weakness of the H-bonding.

Variable temperature experiments of 9, 12 and 15 were mostly in agreement with the observations from the other studies, showing temperature coefficients (Δδ/ΔT) > −3.8 ppb K⁻¹ for all NHs, except for NH2 of compound 9 and NH4 of 12 [(Δδ/ΔT) for NH2 of 9 = −6.5 ppb K⁻¹ and (Δδ/ΔT) for NH4 of 12 = −5.6 ppb K⁻¹] (ESI, Fig. 1, S34†). This suggests that the 15-membered H-bonding in compound 9 is relatively weak, especially at higher temperature. The same is applicable for the NH4 of compound 12.

All of the interesting results found during the solid- and solution-state studies amplified our desire to investigate the conformational features of the oligomers 12 and 15 which could not be crystallized, in spite of the effort put in. This impelled us to undertake nOe-based molecular modeling studies.

Solution state structural investigations

NMR-based structures of compound 12 and 15 were derived from nOe cross peaks by using the Maestro v9.3.518 program from Schrödinger (for a complete table, please refer to the ESI†). The twenty lowest-energy superimposed structures of compounds 12 and 15 showed root mean square deviations (RMSDs) of 0.26 ± 0.06 Å and 0.15 ± 0.06 Å, respectively (Fig. 4). The dynamic ensembled structures of compound 12 revealed that the N-terminus did not reverse as expected when Aib was put in place of Leu. The H-bonding pattern remained the same, as observed for oligomer 9. However, the solution-state structural analysis of compound 15 showed the presence of a second 10-membered ring (C10 H-bonding) between the NH of ^SAnt and the carbonyl of the pivaloyl moiety. Also, the 7-membered ring H-bonding between S=O of ^SAnt and N–H of Leu₂ seen in the previous cases was no longer intact, which was obvious from the DMSO titration studies. The rest of the H-bonding patterns remained the same. Thus, the nOe-based structural investigations concluded that the oligomers display a folded structure.

Fig. 4 Molecular and nOe-derived structures of compounds 12 (a) and 15 (b) showing a folded conformation.

Conclusion

In summary, oligomers carrying ^SAnt as the turn inducer showed compact structures with a folded conformation, which supports the ability of sulfonamides to promote folding. Moreover, all the oligomers containing ^SAnt showed long-range 15-membered ring H-bonding involving four amino acid residues. These results further validate our observations that orthanilic acid (2-aminobenzenesulfonic acid, ^SAnt) is a strong reverse-turn inducer when incorporated into peptide sequences.¹³

Experimental procedures

(S)-Methyl-1-(2-nitrophenylsulfonyl)pyrrolidine-2-carboxylate 1

L-Proline methyl ester hydrochloride (0.82 g, 4.96 mmol) was added to a solution of 2-nitrobenzenesulfonylchloride (1.0 g, 4.51 mmol) in anhy. DCM (10 mL) at 0 °C followed by the addition of Et₃N (1.45 mL, 10.38 mmol). The resulting mixture was then allowed to attain room temperature and was stirred for 12 h. It was then sequentially washed with sat. NaHCO₃, water, dil. HCl and brine. The organic layer was then dried over anhy. Na₂SO₄ and evaporated under reduced pressure to obtain the crude product which on purification by column chromatography furnished 1 as an off-white solid. Yield: 0.81 g (57%); m.p.: 85–86 °C; [α]²⁶_D: −100.0° (c = 1.6, CHCl₃); IR (CHCl₃) ν (cm⁻¹) 3621, 3418, 3020, 1746, 1640, 1546, 1371, 1216, 1163, 770; ¹H NMR (500 MHz, CDCl₃) δ: 8.08 (s, 1H), 7.71–7.69 (m, 2H), 7.63–7.62 (s, 1H), 4.58–4.57 (d, J = 8 Hz, 1H), 3.66 (s, 3H), 3.62–3.60 (m, 1H), 3.55–3.52 (m, 1H), 2.29–2.22 (m, 1H), 2.10–1.94 (m, 3H); ¹³C NMR (125 MHz, CDCl₃) δ: 172.2, 148.0, 133.6, 133.5, 132.7, 132.6, 131.6, 130.9, 124.0, 60.8, 52.3, 48.4, 30.8, 24.4; ESI MS: 337.04 (M + Na)⁺; anal. calcd for C₁₂H₁₄N₂O₆S: C, 45.85; H, 4.49; N, 8.91; found: C, 45.32; H, 4.73; N, 9.28.

(S)-Methyl-1-(2-aminophenylsulfonyl)pyrrolidine-2-carboxylate 2

10% Pd–C (0.015 g) was added to a solution of 1 (0.15 g, 0.47 mmol) in methanol (6 mL). The reaction mixture was then stirred at 60 psi under a hydrogen atmosphere for 8 h, followed by filtration of the catalyst through celite, and the filtrate was evaporated to obtain product 2 which was carried forward without further purification.

(S)-Methyl-1-(2-(2-bromo-2-methylpropanamido)phenylsulfonyl)pyrrolidine-2-carboxylate 3

To a solution of 2 (4.0 g, 14.1 mmol) in dry DCM (25 mL), anhy. Et₃N (2.55 mL, 18.3 mmol) was added at 0 °C followed by the slow addition of 2-bromo-2-methylpropanoyl bromide (1.92 mL, 15.5 mmol). The resulting mixture was then allowed to attain room temperature and was stirred for 12 h, following which it was sequentially washed with sat. NaHCO₃, water and brine. The organic layer was then dried over anhydrous Na₂SO₄ and evaporated under reduced pressure to obtain the crude product which on purification by column chromatography furnished 3 as a viscous liquid. Yield: 5.5 g (90%); [α]²⁶_D: −63.08° (c = 1.3, CHCl₃); IR (CHCl₃) ν (cm⁻¹) 3349, 3020, 1740, 1688, 1588, 1338, 1215, 1154, 759; ¹H NMR (400 MHz, CDCl₃) δ: 10.41 (s, 1H), 8.55–8.53 (d, J = 8 Hz, 1H), 7.91–7.89 (dd, J = 8.0, 1.6 Hz, 1H) 7.61–7.57 (t, J = 7 Hz, 1H), 7.26–7.22 (t, J = 7 Hz, 1H), 4.39–4.36 (dd, J = 8.0, 2.5 Hz, 1H), 3.64–3.59 (m, 4H), 3.49–3.43 (m, 1H), 2.13–2.09 (m, 1H), 2.06 (s, 6H), 2.04–1.98 (m, 2H), 1.93–1.84 (m, 1H); ¹³C NMR (100 MHz, CDCl₃) δ: 171.9, 170.4, 136.5, 134.2, 129.6, 125.8, 124.0, 122.1, 60.4, 59.9, 52.5, 48.4, 31.7, 31.0, 24.5; ESI MS: 455.01 (M + Na)⁺; anal. calcd. for C₁₆H₂₁BrN₂O₅S: C, 44.35; H, 4.88; N, 6.46; found: C, 44.62; H, 5.26; N, 6.08.

Methyl((2-(2-azido-2-methylpropanamido)phenyl)sulfonyl)-L-prolinate 4

Sodium azide (0.68 g, 10.5 mmol) was added to a solution of 3 (1.2 g, 3.5 mmol) in anhy. DMF (10 mL) and the reaction mixture was maintained at 70 °C for 12 h. It was then cooled to room temperature, following which EtOAc (40 mL) was added and the organic layer was washed with water and brine, dried over anhydrous Na₂SO₄ and evaporated under reduced pressure to obtain the crude product which on purification by column chromatography gave 4 as a viscous liquid. Yield: 0.88 g (81%); [α]²⁶_D: −50.53° (c = 0.95, CHCl₃); IR (CHCl₃) ν (cm⁻¹) 3436, 3020, 2120, 1739, 1685, 1585, 1340, 1217, 1140, 770; ¹H NMR (400 MHz, CDCl₃) δ: 10.39 (s, 1H), 8.52–8.50 (d, J = 8 Hz, 1H), 7.89–7.87 (dd, J = 8.0, 1.5 Hz, 1H), 7.58–7.54 (t, J = 7 Hz, 1H), 7.24–7.20 (t, J = 7 Hz, 1H), 4.39–4.36 (dd, J = 8.0, 2.8 Hz, 1H), 3.60–3.54 (m, 4H), 3.46–3.40 (m, 1H), 2.15–2.07 (m, 1H), 2.05–1.95 (m, 1H), 1.93–1.84 (m, 1H), 1.62 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ: 171.9, 171.1, 136.1, 134.1, 129.7, 126.2, 124.0, 122.2, 64.6, 60.2, 52.4, 48.3, 31.0, 24.5; ESI MS: 418.07 (M + Na)⁺; anal. calcd for C₁₆H₂₁N₅O₅S: C, 48.60; H, 5.35; N, 17.71; found: C, 48.29.; H, 5.11.; N, 17.09.

Methyl((2-(2-amino-2-methylpropanamido)phenyl)sulfonyl)-L-prolinate 5

The crude product 5 was obtained from 4, following the procedure mentioned for 2, and it was used for the next step without further purification.

Methyl-((2-(2-(2-((tert-butoxycarbonyl)amino)-4-methylpentanamido)-2-methylpropanamido)phenyl)sulfonyl)-L-prolinate 6a

Representative procedure. EDC·HCl (0.142 g, 0.69 mmol) was added to a solution of 5 (0.17 g, 0.46 mmol) and Boc–Leu–OH (0.12 g, 0.50 mmol) in anhy. DCM at 0 °C, followed by HOBt (0.062 g, 0.46 mmol). The resulting mixture was then stirred at 0 °C for 10 min and at room temperature for 12 h. To the reaction mixture, 30 mL DCM was added and the organic layer was washed sequentially with sat. NaHCO₃, water, sat. KHSO₄ and brine. It was concentrated under reduced pressure and finally purified by column chromatography to furnish a white solid. Yield: 0.24 g (85%); m.p.: 90–92 °C; [α]²⁵_D: −82° (c = 1, CHCl₃); IR (CHCl₃) ν (cm⁻¹) 3337, 3020, 2400, 1700, 1503, 1337, 1215, 1155, 1022, 759, 699; ¹H NMR (400 MHz, CDCl₃) δ: 10.12 (s, 1H), 8.63–8.61 (d, 1H, J = 8.53 Hz), 7.82–7.80 (d, 1H, J = 8.03 Hz), 7.58–7.54 (m, 1H), 7.20–7.16 (m, 1H), 6.93 (bs, 1H), 5.01 (bs, 1H), 4.34–4.31 (m, 1H), 4.19 (bs, 1H), 3.68 (s, 3H), 3.52–3.50 (m, 1H), 3.32–3.26 (m, 1H), 2.08–1.98 (m, 3H), 1.82–1.66 (m, 3H), 1.66 (s, 3H), 1.59 (s, 3H), 1.54–1.48 (m, 1H), 1.45 (s, 9H), 0.94–0.91 (m, 6H); ¹³C NMR (100 MHz, CDCl₃) δ: 174.6, 172.9, 171.5, 137.3, 134.8, 129.8, 123.4, 123.1, 122.7, 79.4, 61.8, 57.4, 56.1, 53.4, 52.4, 49.7, 40.0, 30.9, 28.2, 26.0, 25.0, 24.9, 24.7, 24.6, 24.3, 24.1, 22.9, 22.7, 22.0; LC-MS: 605.25 (M + Na)⁺; anal. calcd. for C₂₇H₄₂N₄O₈S: C, 55.65; H, 7.27; N, 9.62; found: C, 55.81; H, 7.05.; N, 9.75.

Methyl((2-(2-(2-((tert-butoxycarbonyl)amino)-2-methylpropanamido)-2-methylpropanamido)phenyl)sulfonyl)-L-prolinate 10a

Tetramer 10a was obtained as a white fluffy solid. Yield: 90%; m.p.: 67–69 °C; [α]²⁵_D: −12° (c = 1, CHCl₃); IR (CHCl₃) ν (cm⁻¹) 3337, 3019, 2400, 1735, 1699, 1523, 1338, 1215, 1045, 928, 758, 669; ¹H NMR (400 MHz, CDCl₃) δ: 10.04 (s, 1H), 8.58–8.56 (d, 1H, J = 8.54 Hz), 7.82–7.79 (d, 1H, J = 8.03 Hz), 7.56–7.52 (m, 1H), 7.27 (bs, 1H), 7.19–7.15 (m, 1H), 5.03 (s, 1H), 4.33–4.30 (m, 1H), 3.64 (s, 3H), 3.53–3.48 (m, 1H), 3.35–3.29 (m, 1H), 2.11–1.92 (m, 3H), 1.86–1.77 (m, 1H), 1.61 (s, 6H), 1.49 (s, 6H), 1.43 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ: 174.5, 173.2, 172.0, 155.0, 137.1, 134.2, 129.2, 124.8, 123.4, 122.3, 60.3, 57.4, 56.9, 52.4, 48.3, 30.8, 28.2, 25.3, 25.2, 24.9, 24.4; LC-MS: 477.15 (M + Na)⁺, 493.14 (M + K)⁺; anal. calcd. for C₂₅H₃₈N₄O₈S: C, 54.14; H, 6.91; N, 10.10; found: C, 54.43; H, 6.68; N, 10.28.

Methyl((2-(2-methyl-2-((R)-1-pivaloylpyrrolidine-2-carboxamide)propanamido) phenyl)sulfonyl)-L-prolinate 13a

Compound 13a was obtained as a white fluffy solid. Yield: 88%; m.p.: 60–62 °C; [α]²⁵_D: −88° (c = 1, CHCl₃); IR (CHCl₃) ν (cm⁻¹) 3336, 3019, 2400, 1739, 1699, 1585, 1522, 1338, 1215, 1152, 762, 669; ¹H NMR (400 MHz, CDCl₃) δ: 10.08_rotamer (0.2H), 10.04_rotamer (0.8H), 8.57_rotamer (0.2H), 8.56–8.54_rotamer (0.8H), 7.78–7.77 (m, 1H), 7.55_rotamer (0.2H), 7.52_rotamer (0.8H), 7.50–7.49 (m, 1H), 7.15–7.12 (m, 1H), 4.66_rotamer (0.2H), 4.64–4.62_rotamer (0.8H), 4.30–4.28 _rotamer (0.8H), 4.26–4.25 _rotamer (0.2H), 3.67–3.65 (m, 2H), 3.61_rotamer (2H), 3.61_rotamer (1H), 3.50–3.46 (m, 1H), 3.32–3.27 (m, 1H), 2.25–2.22 (m, 1H), 2.04–2.00 (m, 2H), 1.98–1.91 (m, 2H), 1.87–1.77 (m, 3H), 1.55–1.54 (m, 6H), 1.23_rotamer (7H), 1.23_rotamer (2H).; ¹³C NMR (100 MHz, CDCl₃) δ: 178.0, 173.1, 173.0, 171.9, 171.7, 171.6, 137.2, 134.1, 129.2, 129.1, 124.9, 124.8, 123.3, 123.2, 122.2, 122.0, 61.8, 61.6, 60.3, 60.1, 57.4, 52.3, 48.2, 39.1, 30.7, 27.4, 25.8, 25.6, 24.5. 24.4, 23.9; LC MS: 573.23 (M + Na)⁺, 589.20 (M + K)⁺; anal. calcd. for C₂₅H₃₆N₄O₇S: C, 55.95; H, 6.76; N, 10.44; found: C, 55.52; H, 6.99; N, 10.50.

((2-(2-(2-((tert-Butoxycarbonyl)amino)-4-methylpentanamido)-2-methylpropanamido) phenyl)sulfonyl)-L-proline 6b

Representative procedure. LiOH·H₂O (0.06 g, 1.3 mmol) was added to a solution of 8 (0.2 g, 0.34 mmol) in methanol (5 mL), followed by water (1 mL) at 0 °C. After complete consumption of the starting material (4 h), the solvent was evaporated under reduced pressure, and the free acid was generated by treating with sat. NaHSO₄ solution followed by extraction with DCM (2 × 10 mL). Compound 6b obtained after evaporation of the solvent under vacuum was carried forward without further purification.

Methyl-2-(2-((2S)-1-((2-(2-(2-((tert-butoxycarbonyl)amino)-4-methylpentanamido)-2-methylpropanamido)phenyl)sulfonyl)pyrrolidine-2-carboxamido)-4-methylpentanamido)-2-methylpropanoate 8

Representative procedure. A solution of free acid 6b (0.2 g 0.35 mmol) and dimer amine 12 (0.09 g, 0.38 mmol) in anhy. DCM was cooled to 0 °C. To this mixture, EDC·HCl (0.11 g, 0.52 mmol) was added followed by HOBt (0.05 g, 0.35 mmol), and was stirred at 0 °C for 10 min followed by 12 h at room temperature. DCM (30 mL) was then added to the reaction mixture and the organic layer was washed sequentially with sat. NaHCO₃, water, sat. KHSO₄ and brine, concentrated under reduced pressure and finally purified by column chromatography to furnish a white solid. Yield: 0.2 g (75%); m.p.: 115–117 °C; [α]²⁵_D: −101.69° (c = 1.18, CHCl₃); IR (CHCl₃) ν (cm⁻¹) 3394, 3019, 2400, 1674, 1523, 1338, 1215, 1046, 928, 755, 669; ¹H NMR (400 MHz, CDCl₃) δ: 10.17 (s, 1H), 8.52–8.51 (d, 1H, J = 7.93 Hz), 7.83–7.82 (d, 1H, J = 7.93 Hz), 7.61–7.58 (m, 1H), 7.43 (bs, 1H), 7.32 (bs, 1H), 7.23–7.20 (m, 1H), 5.64 (s, 1H), 4.17–4.14 (m, 2H), 3.70 (s, 3H), 3.67 (bs, 1H), 3.26–3.15 (m, 2H), 2.08–2.06 (m, 1H), 1.90 (bs, 1H), 1.84–1.82 (m, 2H), 1.75–1.72 (m, 1H), 1.64 (s, 3H), 1.61 (bs, 1H), 1.59–1.57 (m, 2H), 1.53 (s, 3H), 1.50 (s, 3H), 1.47 (s, 3H), 1.43 (s, 9H), 0.96–0.95 (d, 3H, J = 6.71 Hz), 0.91–0.89 (m, 9H); ¹³C NMR (100 MHz, CDCl₃) δ: 174.7, 173.0, 171.5, 156.3, 137.3, 134.9, 129.8, 123.9, 123.2, 123.0, 79.9, 61.9, 57.5, 56.2, 53.4, 52.4, 49.8, 40.0, 31.0, 28.2, 26.1, 25.1, 24.9, 24.8, 24.7, 24.4, 24.1, 23.0, 22.8, 22.1; LC-MS: 803.40 (M + Na)⁺; anal. calcd. for C₃₇H₆₀N₆O₁₀S: C, 56.90; H, 7.74; N, 10.76; found: C, 56.63.; H, 7.92.; N, 10.68.

Methyl-2-(2-((S)-1-((2-(2-(2-((tert-butoxycarbonyl)amino)-2-methylpropanamido)-2-methylpropanamido)phenyl)sulfonyl)pyrrolidine-2-carboxamido)-4-methylpentanamido)-2-methylpropanoate 11

Hexapeptide 11 was obtained as a white fluffy solid. Yield: 82%; m.p.: 93–95 °C; [α]²⁵_D: −101° (c = 1, CHCl₃); IR (CHCl₃) ν (cm⁻¹) 3393, 3019, 2981, 2401, 1725, 1675, 1523, 1294, 1216, 1155, 1073, 926, 759, 668; ¹H NMR (400 MHz, CDCl₃) δ: 9.97 (s, 1H), 8.51–8.49 (d, 1H, J = 8.24 Hz), 7.81–7.79 (d, 1H, J = 7.33 Hz), 7.61–7.58 (m, 1H), 7.36 (bs, 1H), 7.24–7.21 (m, 1H), 7.08–7.06 (d, 1H, J = 8.55 Hz), 5.20 (s, 1H), 4.46–4.41 (m, 1H), 4.19–4.17 (m, 1H), 3.69 (s, 3H), 3.60 (bs, 1H), 3.18–3.13 (m, 1H), 2.22 (bs, 1H), 2.07–2.04 (m, 1H), 1.91–1.86 (m, 1H), 1.82–1.79 (m, 2H), 1.72–1.71 (m, 1H), 1.60 (s, 3H), 1.58 (s, 3H), 1.54–1.52 (m, 1H), 1.50–1.49 (m, 6H), 1.47 (bs, 6H), 1.42 (s, 9H), 0.94–0.93 (d, 3H, J = 6.41 Hz), 0.91–0.89 (d, 3H, J = 6.41 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 174.8, 174.6, 173.1, 171.1, 170.9, 155.1, 137.3, 134.9, 129.7, 123.9, 123.5, 123.0, 80.2, 77.2, 62.1, 57.3, 56.7, 56.1, 52.3, 51.8, 49.6, 40.3, 30.7, 28.2, 25.2, 25.1, 25.0, 24.6, 24.5, 22.9, 21.6; LC-MS: 775.39 (M + Na)⁺, 791.30 (M + K)⁺; anal. calcd. for C₃₅H₅₆N₆O₁₀S: C, 55.83; H, 7.50; N, 11.16; found: C, 56.03; H, 7.29; N, 11.30.

Methyl-2-methyl-2-(4-methyl-2-(1-((2-(2-methyl-2-((S)-1-pivaloylpyrrolidine-2-carboxamido)propanamido)phenyl)sulfonyl)pyrrolidine-2-carboxamido)pentanamido) propanoate 14

Hexapeptide 14 was obtained as a white fluffy solid. Yield: 73%; m.p.: 98–100 °C; [α]²⁶_D: −129.52° (c = 1.05, CHCl₃); IR (CHCl₃) ν (cm⁻¹) 3333, 3019, 2973, 2400, 1738, 1681, 1584, 1524, 1337, 1215, 1153, 926, 758, 668; ¹H NMR (400 MHz, CDCl₃) δ: 10.03_rotamer (0.8H), 9.96_rotamer (0.2H), 8.52–8.50_rotamer (0.8H), 8.43–8.41_rotamer (0.2H), 7.79–7.77 (d, 1H, J = 7.94 Hz), 7.60–7.56 (m, 2H), 7.22–7.19 (m, 1H), 7.06 (bs, 1H), 6.98–6.96_rotamer (0.8H), 6.94_rotamer (0.2H), 4.66–4.65 (m, 1H), 4.49–4.46 (m, 1H), 4.18–4.17_rotamer (0.2H), 4.15–4.12_rotamer (0.8H), 3.68 (s, 3H), 3.65–3.61 (m, 2H), 3.15–3.10 (m, 1H), 2.20–2.18 (m, 1H), 2.07–2.01 (m, 2H), 1.91–1.76 (m, 5H), 1.70–1.68 (m, 1H), 1.55 (s, 3H), 1.54 (s, 3H), 1.53–1.50 (m, 1H), 1.48 (s, 3H), 1.45 (s, 3H), 1.22_rotamer (7H), 1.19_rotamer (2H), 0.93–0.92 (m, 3H), 0.89–0.88 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 178.1, 174.5, 173.3, 173.2, 172.0, 171.9, 171.1, 171.0, 170.8, 137.5, 134.8, 134.7, 129.6, 124.0, 123.9, 123.8, 123.2, 122.8, 62.3, 62.2, 61.7, 61.6, 57.4, 56.1, 52.3, 52.2, 51.6, 49.4, 48.4, 48.3, 40.3, 40.2, 39.0, 30.8, 30.7, 27.4, 27.3, 25.9, 25.8, 25.6, 25.1, 25.0, 24.9, 24.6, 24.5, 24.0, 21.6, 21.5; LC-MS: 771.41 (M + Na)⁺, 783.38 (M + K)⁺; anal. calcd. for C₃₆H₅₆N₆O₉S: C, 57.73; H, 7.54; N, 11.22; found: C, 57.51; H, 7.38; N, 11.60.

tert-Butyl-(4-methyl-1-((2-methyl-1-((2-(((2S)-2-((4-methyl-1-((2-methyl-1-(methylamino)-1-oxopropan-2-yl)amino)-1-oxopentan-2-yl)carbamoyl)pyrrolidin-1-yl)sulfonyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopentan-2-yl) carbamate 9

Representative procedure. To the ester 9 (0.15 g, 0.02 mmol), a saturated solution of methylamine in methanol was added at 0 °C and stirred for 12 h. The solvent was then removed to obtain the methyl amide 14 as a white solid. Yield: 0.13 g (90%); m.p.: 203–205 °C; [α]²⁵_D: −55.55° (c 1.56, CHCl₃); IR (CHCl₃) ν (cm⁻¹) 3615, 3393, 3019, 2400, 1674, 1523, 1421, 1338, 1215, 1046, 759, 669; ¹H NMR (400 MHz, CDCl₃) δ: 10.15 (s, 1H), 8.63–8.62 (d, 1H, J = 7.93 Hz), 7.79–7.77 (d, 1H, J = 7.93 Hz), 7.72 (s, 1H), 7.61–7.58 (m, 1H), 7.32 (bs, 1H), 7.23–7.20 (m, 1H), 7.04 (s, 1H), 6.77 (s, 1H), 5.24 (bs, 1H), 4.25 (bs, 1H), 4.11 (bs, 1H), 4.05–4.04 (m, 1H), 3.64 (bs, 1H), 3.22–3.20 (m, 1H), 2.72 (s, 3H), 2.09 (bs, 2H), 1.78–1.72 (m, 3H), 1.71–1.64 (m, 3H), 1.61 (s, 3H), 1.57 (s, 3H), 1.51 (s, 3H), 1.46 (bs, 2H), 1.41 (s, 3H), 1.39 (s, 9H), 1.02–1.01 (m, 3H), 0.94–0.90 (m, 9H); ¹³C NMR (100 MHz, CDCl₃) δ: 174.6, 173.4, 173.1, 172.1, 171.9, 155.7, 137.6, 135.0, 129.6, 123.7, 122.9, 122.5, 79.5, 61.4, 57.6, 54.0, 52.6, 50.2, 41.5, 39.5, 30.6, 28.2, 26.5, 25.2, 24.9, 24.5, 24.3, 23.6, 23.0, 22.7, 22.0, 21.7; LC-MS: 802.41 (M + Na)⁺, 818.37 (M + K)⁺; anal. calcd. for C₃₇H₆₁N₇O₉S: C, 56.98; H, 7.88; N, 12.57; found: C, 56.50; H, 8.02; N, 12.89.

tert-Butyl(2-methyl-1-((2-methyl-1-((2-(((2S)-2-((4-methyl-1-((2-methyl-1-(methylamino)-1-oxopropan-2-yl)amino)-1-oxopentan-2-yl)carbamoyl)pyrrolidin-1-yl)sulfonyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl) carbamate 12

Compound 12 was obtained as a white solid. Yield: 82%, m.p.: 136–138 °C; [α]²⁵_D: −63.52° (c = 0.85, CHCl₃); IR (CHCl₃) ν (cm⁻¹) 3627, 3393, 3019, 2400, 1674, 1522, 1422, 1338, 1215, 1046, 928, 757, 669; ¹H NMR (400 MHz, CDCl₃) δ: 9.93 (s, 1H), 8.50–8.49 (d, 1H, J = 8.24 Hz), 7.78–7.77 (m, 1H), 7.63–7.60 (m, 1H), 7.35 (bs, 1H), 7.25–7.20 (m, 2H), 6.82–6.81 (m, 1H), 5.17 (s, 1H), 4.28–4.26 (m, 1H), 4.15–4.13 (m, 1H), 3.66–3.62 (m, 1H), 3.18–3.13 (m, 1H), 2.79–2.76 (m, 3H), 2.06–2.02 (m, 2H), 1.94–1.88 (m, 1H), 1.82–1.79 (m, 2H), 1.75–1.69 (m, 1H), 1.63–1.62 (m, 1H), 1.62 (s, 3H), 1.60 (s, 3H), 1.51 (s, 6H), 1.50 (s, 3H), 1.49 (s, 3H), 0.98–0.97 (d, 3H, J = 6.41 Hz), 0.93–0.92 (d, 3H, J = 6.41 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 174.9, 174.7, 173.2, 172.1, 171.3, 155.0, 137.3, 134.9, 129.6, 124.0, 123.7, 122.9, 80.2, 77.2, 62.1, 57.5, 57.4, 56.7, 53.1, 49.6, 39.3, 30.7, 28.2, 26.5, 25.7, 25.5, 25.4, 25.3, 25.1, 24.6, 24.5, 22.9, 21.6; LC-MS: 774.45 (M + Na)⁺, 790.43 (M + K)⁺; anal. calcd. for C₃₅H₅₇N₇O₉S: C, 55.91; H, 7.64; N, 13.04; found: C, 56.13; H, 7.45; N, 13.28.

(2S)-N-(2-Methyl-1-((2-(((2S)-2-((4-methyl-1-((2-methyl-1-(methylamino)-1-oxopropan-2-yl)amino)-1-oxopentan-2-yl)carbamoyl)pyrrolidin-1-yl)sulfonyl)phenyl)amino)-1-oxopropan-2-yl)-1-pivaloylpyrrolidine-2-carboxamide 15

Compound 15 was obtained as a white solid. Yield: 86%; m.p.: 162–164 °C; [α]²⁵_D: −104° (c = 1, CHCl₃); IR (CHCl₃) ν (cm⁻¹) 3615, 3393, 3019, 2400, 1674, 1523, 1421, 1338, 1215, 1046, 928, 767, 669; ¹H NMR (400 MHz, CDCl₃) δ: 9.94_rotamer (0.2H), 9.91_rotamer (0.8H), 8.43–8.42 (d, 1H, J = 8.31 Hz), 7.79–7.77_rotamer (0.9H), 7.76_rotamer (0.7H), 7.67_rotamer (0.7H), 7.64_rotamer (0.3H), 7.61–7.58 (m, 1H), 7.25–7.22 (m, 1H), 7.07–7.04 (m, 2H), 6.92 (bs, 1H), 4.65–4.62 (m, 1H), 4.35–4.30_rotamer (0.8H), 4.28–4.26_rotamer (0.2H), 4.14–4.13_rotamer (0.1H), 4.12–4.10_rotamer (0.9H), 3.72–3.59 (m, 3H), 3.16–3.11 (m, 1H), 2.78–2.77_rotamer (2.5H), 2.76_rotamer (0.5H), 2.19–2.14 (m, 1H), 2.12–2.08 (m, 1H), 2.04–1.96 (m, 3H), 1.93–1.87 (m, 2H), 1.82–1.77 (m, 2H), 1.73–1.69 (m, 1H), 1.63–1.58 (m, 1H), 1.55 (s, 3H), 1.54 (s, 3H), 1.53 (s, 3H), 1.50 (s, 3H), 1.20_rotamer (2H), 1.16_rotamer (7H), 0.99–0.97 (m, 3H), 0.93–0.91 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ: 178.1, 177.8, 174.9, 174.7, 172.3, 172.2, 172.2, 172.0, 171.7, 137.7, 137.6, 134.9, 129.7, 129.5, 124.3, 124.2, 124.1, 123.4, 61.9, 61.7, 57.6, 57.5, 57.3, 53.1, 52.9, 49.6, 48.3, 40.0, 39.1, 39.0, 30.9, 27.3, 26.5, 25.8, 25.7, 25.6, 25.5, 24.3, 25.1, 25.0, 24.6, 24.5, 23.0, 22.9, 21.6, 21.5; LC-MS: 770.47 (M + Na)⁺, 786.41 (M + K)⁺; anal. calcd. for C₃₆H₅₇N₇O₈S: C, 57.81; H, 7.68; N, 13.11; found: C, 57.55; H, 7.89; N, 13.35.

Acknowledgements

AR, ASK and RLG are thankful to CSIR, New Delhi, for a research fellowship. GJS thanks NCL-IGIB (New Delhi) for financial support.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: ¹H, ¹³C, DEPT-135 NMR, 2D study spectra, ESI mass spectra and theoretical study of new compounds are included. CCDC [927811]. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c3ra47039c

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