Synthesis and Analysis of Macrocyclic Peptides with 3₁₀-Helical Structure

Abstract

Macrocyclic peptides were designed and synthesized for secondary structural analysis. A PyBroP-based cyclization protocol was employed and was facilitated by solid-phase synthesis. After systematic NMR analyses of each macrocycle, multiple structures were found to exist as 3₁₀-helices.

Key words

peptide - macrocycle - PyBroP - helix - secondary structure

• References and Notes:

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• 8 Synthesis of 9a Knorr amide MBHA resin (0.50 g, 0.15 mmol) was swelled in DMF for 2 h. The suspension was filtered, and 20% piperidine in DMF (3 resin volumes) was added into the resin to cleave the Fmoc group. The suspension was kept at r.t. for 0.5 h while a stream of nitrogen was bubbled through it. After 0.5 h, the suspension was filtered and washed with DMF (5 × 3 resin volumes). The suspension was filtered, and Fmoc-Dab(Boc)-OH (263 mg, 0.6 mmol), HBTU (216 mg, 0.57 mmol), NMM (132 μL, 1.2 mmol), and DMF (about 1.2 resin volumes) were added to the resin. The reaction was carried out under a nitrogen atmosphere. Kaiser ninhydrin test was used to indicate reaction completion. After the reaction was complete, the suspension was filtered and washed with DMF (5 × 3 resin volumes). Then, 20% piperidine in DMF (3 resin volumes) was added into the resin to cleave the Fmoc group. The suspension was kept at r.t. for 0.5 h while a stream of nitrogen was bubbled through it. After 0.5 h, the suspension was filtered and the resin was washed with DMF (5 × 3 resin volumes). Subsequent amide couplings followed the generic procedure. Fmoc-Xaa (4 equiv), HBTU (3.8 equiv), and NMM (8 equiv) in DMF were added to the resin. The reaction mixture was agitated under nitrogen until the amidation went to completion based on the Kaiser ninhydrin test. After the reaction was deemed complete, the suspension was filtered, and the resin was washed with DMF (5 × 3 resin volumes). Then, 20% piperidine in DMF (3 resin volumes) was added to the resin-bound peptide. The suspension was kept at r.t. for 0.5 h while a stream of nitrogen was bubbled through it. After 0.5 h, the suspension was filtered, and the resin was washed with DMF (5 × 3 resin volumes). This procedure was repeated until 4-PyrAla N-oxide-Ala-Leu-Ala-Dab-Knorr amide MBHA resin-bound peptide was delivered. A mixture of Ac₂O–NMM–DMF (1.7:1:14, 3 resin volumes) was then added to 4-PyrAla-N-oxide-Ala-Leu-Ala-Dab-Knorr amide MBHA resin-bound peptide, and the reaction mixture was kept under nitrogen. The reaction was deemed complete based on the Kaiser ninhydrin test to deliver Ac-(4-PyrAla-N-oxide)-Ala-Leu-Ala-Dab-Knorr amide MBHA resin-bound peptide. The peptidyl resin was then washed with DMF (5 × 10 mL) and MeOH (5 × 10 mL) and dried in vacuo overnight. The peptidyl resin was then treated with a mixture of TFA–H₂O (95:5), and the mixture was kept at r.t. for 3 h, and then filtered. Et₂O (7–8 filtrate volumes) was added to the filtrate which resulted in a precipitate. The mixture was centrifuged, and the supernatant was decanted. The resulting pellet was then washed with Et₂O (3 × 7–8 filtrate volume) and dried under vacuum overnight to afford crude Ac-(4-PyrAla N-oxide)-Ala-Leu-Ala-Dab-NH₂ (5). THF (5 mL) was added to the crude peptide followed by PyBroP (200 mg, 0.45 mmol) and DIPEA (130 μL, 0.75 mmol). The reaction mixture was then stirred at r.t. for 5 h, whereupon it was concentrated under reduced pressure to give crude 9a. The crude peptide (pellet) was purified by reverse-phase HPLC described in the Supporting Information (purification method 1) using a solvent gradient of A/B (90:10 to 30:70) over 60 min at a flow rate of 20 mL/min. Like fractions were combined, and the peptide was repurified by eluting with a solvent gradient of A/B (85:15 to 65:35) over 60 min at a flow rate of 20 mL/min. Like fractions were once again combined and lyophilized to deliver 8 mg (9.5%) of 9a as a white solid. UV purity (220 nm) = 96% (t _R = 8.86 min; solvent gradient A/C = 82:18 to 72:28); ESI-MS: m/z = 1121.2 [M + H]⁺, 561.4 [M/2 + H]⁺. ¹H NMR (600 MHz, DMSO-d ₆, 37 °C): δ = 8.39 (d, J = 7.8 Hz, 1 H), 8.34 (d, J = 7.7 Hz, 1 H), 8.27 (d, J = 4.9 Hz, 1 H), 8.03 (d, J = 8.7 Hz, 1 H), 7.99 (br s, 1 H), 7.89 (d, J = 6.5 Hz, 1 H), 7.22 (dd, J = 7.9, 4.9 Hz, 2 H), 7.10 (br s, 1 H), 7.08 (s, 1 H), 6.71 (d, J = 6.5 Hz, 1 H), 4.56 (q, J = 6.8 Hz, 1 H), 4.38–4.22 (m, 2 H), 4.21–4.00 (m, 2 H), 3.34 (d, J = 6.7 Hz, 2 H), 3.15–3.02 (m, 1 H), 2.89 (dd, J = 14.9, 6.8 Hz, 1 H), 2.13–1.98 (m, 1 H), 1.88 (s, 3 H), 1.83–1.72 (m, 1 H), 1.66–1.52 (m, 1 H), 1.45 (ddd, J = 13.5, 8.8, 4.7 Hz, 1 H), 1.37 (ddd, J = 13.6, 10.1, 5.1 Hz, 1 H), 1.25 (d, J = 7.1 Hz, 3 H), 1.22 (d, J = 7.3 Hz, 3 H), 0.88 (t, J = 6.3 Hz, 6 H).
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The 3₁₀-helix is the fourth most common type of secondary structure in proteins, see:
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• 17 See the Supporting Information for synthetic details.