Rapid and Efficient Methodology to Perform Macrocyclization Reactions in Solid-Phase Peptide Chemistry

Abstract

Solid-phase peptide synthesis and microwave-assisted organic synthesis have been combined in order to speed up drug discovery process of macrocyclization products in high purity and good yield.

solid-phase peptide synthesis - macrocyclization - ­microwave synthesis

References

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General Procedure. All experiments were carried out in a Milestone CombiChem Microwave Synthesizer with vessels of 4 mL volume, using DMF as solvent. In all irradiation experiments, rotation of the rotor, irradiation time, temperature, and power were monitored with the ‘easyWAVE’ software package. Temperature was monitored with the aid of an optical fiber inserted into one of the reaction containers. Once 50 °C was reached the reaction mixture was held at this temperature for 10 min and then cooled rapidly to r.t. The reaction vessels were opened and the contents were poured into a separating funnel. The compounds were washed and subjected to the final cleavage from the resin. The crude product was precipitated by anhyd diethyl ether and recovered by filtration. All final products were purified by reverse-phase HPLC carried out on Vydac C-18 column with the following dimensions: 25 × 0.46 cm for analysis and 25 × 2.2 cm for preparative work. Flow rate applied were 1.0 mL/min (C₁₈ column, linear gradient 10-90% MeCN/H₂O in 40 min) and 5.0 mL/min for analytical and preparative HPLC, respectively. Analysis of purified products was performed by reverse-phase HPLC, ¹H NMR and mass spectroscopy.

<A NAME="RG15403ST-11">11</A>

Compound 1: C₄₅H₄₈N₈O₁₀S, M.W.: 892.98. MS (ESI, EI⁺): m/z = 892.7 (M⁺). HPLC: k’ = 5.85. ¹H NMR (500 MHz, DMSO-d ₆): δ = 10.48 (s, 1 H), 9.14 (s, 1 H), 8.97 (d, 1 H), 8.29 (d, 1 H), 8.22 (d, 1 H), 8.04 (d, 1 H), 7.86 (s, 1 H), 7.80 (d, 1 H), 7.68 (d, 1 H), 7.58 (m, 2 H), 7.33 (d, 1 H), 7.27-7.16 (m, 6 H), 7.07 (t, 1 H), 6.99 (t, 1 H), 6.88 (d, 2 H), 6.59 (d, 2 H), 4.52 (m, 1 H), 4.46 (m, 1 H), 4.43 (m, 1 H), 4.28 (m, 1 H), 3.83 (m, 1 H), 3.41 (m, 1 H), 3.30 (m, 2 H), 3.13 (dd, 1 H), 2.99 (q, 1 H), 2.92 (dd, 1 H), 2.79 (m, 1 H), 2.63 (m, 2 H), 1.56 (m, 2 H), 1.48 (m, 1 H), 1.34 (q, 1 H), 1.23 (m, 1 H), 1.06 (m, 1 H). Compound 2: C₄₅H₅₅N₉O₁₀, M.W.: 918.00. MS (ESI, EI⁺): m/z = 918.2 (M⁺). HPLC: k’ = 5.93. ¹H NMR (500 MHz, DMSO-d ₆): δ = 10.87 (s, 1 H), 9.35 (s, 1 H), 8.91 (d, 1 H) 8.38 (d, 1 H), 8.18 (d, 1 H), 8.01 (d, 1 H), 7.57 (m, 2 H), 7.47 (d, 1 H), 7.22-7.16 (m, 3 H), 7.15-7.04 (m, 5 H), 7.00 (m, 1 H), 6.98 (t, 1 H), 6.76 (d, 2 H), 6.65 (d, 2 H), 4.38 (m, 1 H), 4.31 (m, 1 H), 4.22-4.18 (m, 3 H), 3.23 (m, 2 H), 3.16 (m, 1 H), 3.10 (m, 1 H), 2.95 (m, 1 H), 2.84 (m, 1 H), 2.67 (m, 1 H), 2.62-2.59 (m, 3 H), 1.70 (m, 1 H), 1.56-1.53 (m, 2 H), 1.46-1.44 (m, 3 H), 1.37-1.36 (m, 4 H), 1.16-1.12 (m, 3 H), 1.06-1.03 (m, 1 H). Compound 3: C₄₅H₄₉N₉O₉S, M.W.: 891.99. MS (ESI, EI⁺): m/z = 891.3 (M⁺). HPLC: k’ = 5.78. Compound 4: C₄₈H₅₆N₁₀O₉, M.W.: 917.02. MS (ESI, EI⁺): m/z = 917.4 (M⁺). HPLC: k’ = 5.88.

<A NAME="RG15403ST-12">12</A>

All reactions were performed on a scale of 100 mg of beads (0.7 mmol/g) allowing the isolation of at least 10 mg of crude product.

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