A Method for the Generation of Pam₂Cys-Based Lipopeptide Mimics via CuAAC Click Chemistry

Abstract

The immunostimulatory activity of naturally occurring lipopeptides as bacterial cell wall components arises due to the presence of certain lipid motifs. S-[2,3-bis(palmitoyloxy)propyl]cysteine (Pam₂Cys) is a lipid motif known to successfully reproduce this immunostimulatory activity when incorporated into synthetic peptides. Herein we report the synthesis of a Pam₂Cys analogue designed to facilitate the fully convergent, efficient generation of lipopeptide mimics using click chemistry to effect its ligation to suitably functionalized peptides. The incorporation of this Pam₂Cys mimic into peptides prepared by SPPS is also demonstrated.

Key words

lipopeptides - CuAAC - click chemistry - Pam₂Cys

• References and Notes

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• 14 tert-Butyl (2R,2S)-2-Azido-3-(2,3-dihydroxypropane-thio)propanoate (3) To a solution of 1 (1.64 g, 3.46 mmol) in CH₂Cl₂ (10 mL) was added piperidine (3.42 mL, 34.6 mmol). After stirring for 1.5 h at r.t., the solvent was removed in vacuo. The crude product was partially purified by flash chromatography (CH₂Cl₂ → 85:15 CH₂Cl₂–MeOH). Imidazole-1-sulfonylazide·HCl (0.870 g, 4.15 mmol) was added portionwise to an ice-cooled mixture of K₂CO₃ (0.813 g, 5.88 mmol), CuSO₄·5H₂O (9 mg, 34.6 μg), and the crude product in dry MeOH (20 mL). The mixture was allowed to warm to r.t. and after stirring for 6 h under N₂, the solvent was removed in vacuo. H₂O (20 mL) was added to the residue, and the product was extracted with EtOAc (3 × 15 mL). The organic layers were combined, dried with MgSO₄, and concentrated in vacuo. The crude product was purified by silica gel flash chromatography (n-hexanes–EtOAc = 67:33) to afford 3 as a yellow oil (0.475 g, 50% over 2 steps). IR (film): ν_max = 3408.4 (br, alcohol OH), 2928.9 (w, alkane CH), 2114.1 (s, N₃), 1735.7 (s, ester C=O) cm^–1. [α]_D ²⁰ –1.00 (c 1.01, CHCl₃). ¹H NMR (400 MHz, CDCl₃): δ = 4.02 (1 H, t, J = 5.6 Hz, CH), 3.84 (1 H, m, CH), 3.71 (3 H, m, CH₂, OH), 3.21 (1 H, br s, OH), 2.92 (2 H, m, CH₂) 2.74 (2 H, m, CH₂), 1.52 (9 H, s, 3 × CH₃). ¹³C NMR (100 MHz, CDCl₃): δ = 168.0 (CO₂H), 83.8 (C), 71.0 (CH), 65.2 (CH₂), 62.9 (CH), 36.3 (CH₂), 33.8 (CH₂), 28.0 (3 × CH₃). HRMS (ESI⁺): m/z calcd for C₁₀H₁₉N₃NaO₄S⁺ [M + Na⁺]: 300.0988; found: 300.0995
• 15 tert-Butyl (2R,2S)-2-Azido-3-[2,3-bis(palmitoyloxy)-propanethio]propanoate (4) Compound 4 was afforded as a very hygroscopic colourless solid (0.76 g, 74%). [α]_D ²⁰ –6.31 (c 1.01, CHCl₃). IR (film): ν_max = 2923.3 (s, alkane CH), 2853.5 (m, alkane CH), 2114.7 (m, N₃), 1740.8 (s, ester C=O) cm^–1. ¹H NMR (400 MHz, CDCl₃): δ = 5.16 (1 H, m, CH), 4.35 (1 H, dd, J = 4.2, 3.6 Hz, CH₂), 4.19 (1 H, dd, J = 5.6, 3.0 Hz, CH₂), 3.98 (1 H, dt, J = 6.4, 1.2 Hz, CH), 2.87 (4 H, m, 2 × CH₂), 2.31 (4 H, sext, Pam-CH₂), 1.62 (br s, 4 H, Pam-CH₂), 1.51 (9 H, s, 3 × CH₃), 1.26 (48 H, br s, Pam-CH₂), 0.88 (6 H, t, J = 6.8 Hz, Pam-CH₃). ¹³C NMR (100 MHz, CDCl₃): δ = 173.3, 173.0 (2 × CO₂H), 167.7 (CO₂H), 83.6 (C), 70.3 (CH), 63.4 (CH₂), 62.9 (CH), 34.3, 34.1, 33.8 (Pam-CH₂), 33.2 (CH₂), 31.9 (CH₂), 29.7–29.4 (Pam-CH₂), 28.0 (3 × CH₃), 24.9 (Pam-CH₂), 22.7 (Pam-CH₂), 14.1 (Pam-CH₃). MS (ESI⁺): m/z (%) = 777 (25) [MNa⁺], 442 (100). HRMS (ESI⁺): m/z calcd for C₄₂H₇₉N₃NaO₆S⁺ [M + Na]⁺: 776.5582; found: 776.5544
• 16 (2R,2S)-2-Azido-3-[2,3-bis(palmitoyloxy)propanethio]-propanoic Acid (1) Following lyophilization from H₂O–MeCN (1:1) + 0.1% TFA, 1 was afforded as a powdery white solid (547 mg, 84%); mp 37–39 °C; [α]_D ²⁰ –6.08 (c 1.00, CHCl₃). IR (film): ν_max = 2919.1 (s, alkane CH), 2851.2 (m, alkane CH), 2120.0 (m, N₃), 1742.2 (s, C=O ester), 1241.0 (m, acid CO) cm^–1. ¹H NMR (400 MHz, CDCl₃): δ = 9.71 (1 H, br s, CO₂H), 5.18 (1 H, tt, J = 9.2, 3.2 Hz, CH), 4.37 (1 H, dd, J = 8.4, 3.6 Hz, CH₂), 4.21 (2 H, m, CH, CH₂), 3.05 (1 H, m, CH₂), 2.88 (3 H, m, 1.5 × CH₂), 2.33 (4 H, dt, J = 7.2, 5.2 Hz, Pam-CH₂), 1.62 (4 H, m, Pam-CH₂), 1.26 (48 H, br s, Pam-CH₂), 0.88 (6 H, t, J = 6.8 Hz, Pam-CH₃). ¹³C NMR (100 MHz, CDCl₃): δ = 173.7, 173.5 (2 × CO₂H), 173.2 (CO₂H), 70.3 (CH), 63.6 (CH₂), 62.3 (CH), 34.3, 33.7, 33.1 (Pam-CH₂), 33.1 (CH₂), 31.9 (CH₂), 29.7–29.1 (Pam-CH₂), 24.9 (Pam-CH₂), 22.7 (Pam-CH₂), 14.1 (Pam-CH₃). HRMS (ESI⁺): m/z calcd for C₃₈H₇₁N₃NaO₆S⁺ [M + Na]⁺: 720.4956; found: 720.4927

4-Pentynoyl-SKKKK Peptide (5)
• 17a SPPS of the target peptide was undertaken using Fmoc-Lys(Boc)-WANG resin (0.142 g, 0.7 mmol/g loading, 0.1 mmol scale) using microwave-enhanced Fmoc-SPPS as described in: Harris P, Williams G, Shepherd P, Brimble M. Int. J. Pept. Res. Ther. 2008; 14: 387
• 17b Coupling of the N-terminal 4-pentynoyl moiety was undertaken with a coupling mixture of 4-pentynoic acid (101 mg, 1.03 mmol, 10 equiv), HBTU (343 mg, 0.90 mmol, 9 equiv), and i-Pr₂NEt (345 μL, 2.00 mmol, 20 equiv) in DMF (1.0 mL) under microwave irradiation (25 W, 73 °C, 20 min). Following final Fmoc deprotection, the resin was washed with DMF and CH₂Cl₂ and was air dried. Cleavage from the resin was performed by addition of a cleavage cocktail of TFA–H₂O–i-Pr₃SiH (95:2.5:2.5% v/v, 3.0 mL) to the resin and shaking for 2 h. TFA was removed by a flow of N₂, and the crude peptide residue was lyophilised from H₂O–MeCN (1:1 v/v) + 0.1% TFA to afford a white solid (67 mg, 73% purity by HPLC). No further purification was undertaken
• 18 A mixture of azide 1 (4.85 mg, 6.9 μmol), peptide 5 (6.3 mg, 9.0 μmol), CuI·P(OEt)₃ (16.1 mg, 45.1 μmol), and i-Pr₂NEt (15.5 μL, 89.3 μmol) in DMF (2.32 mL, degassed with N₂) was allowed to stand at r.t. under a N₂ atmosphere. After 30 min, no further change was observed by RP HPLC using a Vydac diphenyl 10μ 300 Å 10 × 250 mm analytical column with a 0.5 mL/min flow rate. A gradient of 10% B to 100% B over 20 min was used: buffer A = 0.1% aq TFA; buffer B = MeCN + 0.1% TFA
• 19 Lee DJ, Harris PW. R, Brimble MA. Org. Biomol. Chem. 2011; 9: 1621
• 20 A reaction mixture of azide 1 (1.0 mg, 1.4 μmol), peptide 6 (2.0 mg, 1.0 μmol), CuI·P(OEt)₃ (2.5 mg, 7.0 μmol), and i-Pr₂NEt (2.5 μL, 14.4 μmol) in DMF (365 μL, degassed with N₂) was allowed to stand at r.t. under an N₂ atmosphere. After 30 min, no further change was observed by RP HPLC using a Phenomenex Jupiter C4 5μ 300 Å 2.0 × 50 mm analytical column with a 0.5 mL/min flow rate. A gradient of 10% B to 100% B over 20 min was used: buffer A = 0.1% aq TFA; buffer B = MeCN + 0.1% TFA