Synlett 2012; 23(11): 1617-1620
DOI: 10.1055/s-0031-1291148
letter
© Georg Thieme Verlag Stuttgart · New York

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

Ho Yeung
School of Chemical Sciences, The University of Auckland, 23 Symonds Street, Auckland 1142, New Zealand, Email: [email protected]
,
Dong Jun Lee
School of Chemical Sciences, The University of Auckland, 23 Symonds Street, Auckland 1142, New Zealand, Email: [email protected]
,
Geoffrey M. Williams
School of Chemical Sciences, The University of Auckland, 23 Symonds Street, Auckland 1142, New Zealand, Email: [email protected]
,
Paul W. R. Harris
School of Chemical Sciences, The University of Auckland, 23 Symonds Street, Auckland 1142, New Zealand, Email: [email protected]
,
Rod P. Dunbar
School of Chemical Sciences, The University of Auckland, 23 Symonds Street, Auckland 1142, New Zealand, Email: [email protected]
,
Margaret A. Brimble*
School of Chemical Sciences, The University of Auckland, 23 Symonds Street, Auckland 1142, New Zealand, Email: [email protected]
› Author Affiliations
Further Information

Publication History

Received: 13 March 2012

Accepted: 09 April 2012

Publication Date:
11 June 2012 (online)


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.

 
  • References and Notes

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  • 11 Fmoc deprotection with 20% v/v piperidine in CH2Cl2 or 20% v/v Et2NH in CH2Cl2 followed by diazotransfer with imidazole-1-sulfonyl azide·HCl (5 equiv), K2CO3 (8 equiv), and CuSO4·5H2O (1 mol%) in MeOH
  • 12 Pattabiraman VR, McKinnie SM. K, Vederas JC. Angew. Chem. Int. Ed. 2008; 47: 9472
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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): νmax = 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): νmax = 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): νmax = 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