Synlett 2013; 24(14): 1835-1841
DOI: 10.1055/s-0033-1339349
letter
© Georg Thieme Verlag Stuttgart · New York

An Improved Method for the Synthesis of Lipopeptide TLR2-Agonists Using Click Chemistry

Tom H. Wright
a   School of Chemical Sciences, The University of Auckland, 23 Symonds Street, Auckland 1142, New Zealand   Fax: +64(9)3737422   Email: m.brimble@auckland.ac.nz
,
Anna E. S. Brooks
b   School of Biological Sciences, The University of Auckland, 3a Symonds Street, Auckland 1142, New Zealand
c   Maurice Wilkins Centre, The University of Auckland, 3a Symonds Street, Auckland 1142, New Zealand
,
Alicia J. Didsbury
b   School of Biological Sciences, The University of Auckland, 3a Symonds Street, Auckland 1142, New Zealand
,
Julie D. McIntosh
b   School of Biological Sciences, The University of Auckland, 3a Symonds Street, Auckland 1142, New Zealand
,
Kristina Burkert
b   School of Biological Sciences, The University of Auckland, 3a Symonds Street, Auckland 1142, New Zealand
,
Ho Yeung
b   School of Biological Sciences, The University of Auckland, 3a Symonds Street, Auckland 1142, New Zealand
,
Geoffrey M. Williams
a   School of Chemical Sciences, The University of Auckland, 23 Symonds Street, Auckland 1142, New Zealand   Fax: +64(9)3737422   Email: m.brimble@auckland.ac.nz
,
P. Rod Dunbar
b   School of Biological Sciences, The University of Auckland, 3a Symonds Street, Auckland 1142, New Zealand
c   Maurice Wilkins Centre, The University of Auckland, 3a Symonds Street, Auckland 1142, New Zealand
,
Margaret A. Brimble*
a   School of Chemical Sciences, The University of Auckland, 23 Symonds Street, Auckland 1142, New Zealand   Fax: +64(9)3737422   Email: m.brimble@auckland.ac.nz
c   Maurice Wilkins Centre, The University of Auckland, 3a Symonds Street, Auckland 1142, New Zealand
› Author Affiliations
Further Information

Publication History

Received: 07 June 2013

Accepted: 11 June 2013

Publication Date:
26 July 2013 (online)


Abstract

S-[2,3-Bis(palmitoyloxy)propyl]cysteine (Pam2Cys) is a synthetic lipid motif known to act as a TLR2 agonist when incorporated into peptide vaccines. Herein, we report the synthesis of Pam2Cys-containing, propargyl-functionalised ‘clickable’ building blocks and their conjugation with azide-functionalised antigenic peptides using click chemistry. This method facilitates the fully convergent preparation of self-adjuvanting lipopeptides for use as vaccines.

 
  • References and Notes

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  • 19 General method of peptide synthesis: Peptido-resin was transferred to the reaction vessel of a Tribute automated peptide synthesiser. Automated synthesis was undertaken with cycles of Fmoc deprotection and Fmoc-AA-OH coupling steps. Deprotection was undertaken by addition of 20% v/v piperidine in DMF (3.0 mL) and agitation (2 × 7 min). Following resin drainage and DMF washing (3 × 4 mL), a coupling step was performed with Fmoc-AA-OH (5 equiv) dissolved in HBTU (0.24 mM in DMF, 2 mL). N-Methylmorpholine (2 M in DMF, 0.5 mL) was utilised in the base-addition step. Coupling proceeded for 1 h. After DMF washing steps, the next cycle of deprotection and coupling commenced, repeating until all amino acids were coupled. The resin was then drained, washed with DMF and CH2Cl2 and air-dried. A cleavage cocktail of TFA/H2O/DODT/(i-Pr)3SiH (94:2.5:2.5:1% v/v, 10.0 mL) was added to the anhydrous resin and the mixture was shaken at r.t. for 4 h. The cleavage cocktail was then treated with cold Et2O and triturated with hexane to precipitate the crude peptide, which was centrifuged at 4000 rpm for 5 min. The supernatant was discarded and the pellet was washed with Et2O, before repeating the spinning step. The ether phase was then discarded and the peptide was dried with N2 flow. The crude peptide was then lyophilised from MeCN–H2O (1:1 containing 0.1% TFA). Analytical data for peptide click building blocks Compound 4: R t = 16.10 min (Phenomenex Gemini C18; 5 μm; 110 Å; 2.0 × 50 mm column using a 1–90% MeCN–H2O+0.1% TFA, 4% MeCN per min gradient); MS (ESI+): m/z [M + H]+ calcd for C70H132N12O12S+: 1365.94; found: 1366.8. Compound 5: R t = 13.16 (Phenomenex Gemini C18; 5 μm; 110 Å; 2.0 × 50 mm column using a 1–100% MeCN–H2O+0.1% TFA, 4% MeCN per min gradient); MS (ESI+): m/z [M + H]+ calcd for C72H134N12O13S+: 1407.97; found: 1408.8. Compound 6: R t = 18.40 min (Phenomenex Gemini C18; 5 μm; 110 Å; 2.0 × 50 mm column using a 1–90% MeCN–H2O+0.1% TFA, 4% MeCN per min gradient); MS (ESI+): m/z [M + H]+ calcd for C44H75N13O13S+: 1026.21; found: 1026.6. Compound 7: R t = 14.60 min (Phenomenex Gemini C18; 5 μm; 110 Å; 2.0 × 50 mm column using a 1–90% MeCN–H2O+0.1% TFA, 4% MeCN per min gradient); MS (ESI+): m/z [M + H]+ calcd for C50H87N15O14S+: 1154.38; found: 1154.5. Typical Click Reaction: To the propargylated Pam2CysSK4 4 (2.04 mg, 1.77 μmol) and azido-containing peptide 6 (2.5 mg, 1.77 μmol) in degassed DMSO (500 μL) was added stock solutions of CuSO4·5H2O (0.1 M, 142 μL, 0.0142 mmol) and sodium ascorbate (0.25 M, 57 μL, 0.0142 mmol) in H2O. A yellow-brownish colour indicated reduction of Cu(II) to the desired Cu(I). The mixture was then agitated at 70 °C for 10 min, after which no further change in the reaction mixture could be observed by RP HPLC (Phenomonex C18 Gemini; 110 Å; 5 μm; 4.6 × 250 mm, gradient 1–91% MeCN–H2O+0.1% TFA, 3% MeCN per minute). The crude product 8 was purified by RP HPLC (Phenomenex Gemini C18; 110 Å; 5 μm; 4.6 × 250 mm analytical column; 20–100% MeCN–H2O+0.1% TFA, 3% MeCN per min, 50 °C). Mass spectrometry confirmed the structure of the desired product. Analytical Data for Click Products
    Compound 8:
    R t = 12.70 min (Phenomenex Gemini C18; 5 μm; 110 Å; 2.0 × 50 mm column using a 1–100% MeCN–H2O+0.1% TFA, 4% MeCN per min gradient); MS (ESI+): m/z [M+2H]2+ calcd for C113H205N25O25S2 +: 1195.6; found: 1196.0. Compound 9: R t = 13.8 min (Phenomenex Gemini C18; 5 μm; 110 Å; 2.0 × 50 mm column using a 1–100% MeCN–H2O+0.1% TFA, 4% MeCN per min gradient); MS (ESI+): m/z [M+2H]2+ calcd for C120H219N27O26S2 +: 1260.16; found: 1259.4. Compound 10: R t = 13.80 min (Phenomenex Gemini C18; 5 μm; 110 Å; 2.0 × 50 mm column using a 1–100% MeCN–H2O+0.1% TFA, 4% MeCN per min gradient); MS (ESI+): m/z [M+2H]2+ calcd for C116H209N25O26S2 +: 1217.09; found: 1216.9. Compound 11: R t = 12.70 min (Phenomenex Gemini C18; 5 μm; 110 Å; 2.0 × 50 mm column using a 1–100% MeCN–H2O+0.1% TFA, 4% MeCN per min gradient); MS (ESI+): m/z [M+2H]2+ calcd for C122H221N27O27S2 +: 1281.18; found: 1280.4. Bioassay: A whole blood (WB) sample (100 μL) was incubated with 100 nM, 1 μM and 10 μM of each compound, in duplicate, and incubated overnight at 37 °C in a 5% CO2 humidified incubator. Pam3CSK4 (10 μM; EMC Microcollections) was used as a positive control. To detect activation of monocytes, WB samples were stained with anti-CD14-FITC, anti-HLA-DR-Alexa700, anti-CD80-PE-Cy7, anti-CD40-PE, anti-CD86-APC, or anti-CD16-APC-Cy7 (all from Biolegend) for 20 min at r.t., protected from light. Following incubation, BD FACS lyse (2 mL) was added, incubated for 15 min at r.t., then washed twice with ice-cold wash buffer (PBS, 1% Human Serum). Data acquisition was performed with a BD FACS Aria II (Becton Dickinson) and analysed by using FlowJo software version 7.6.5 (TreeStar). CD80 receptor expression on monocytes was detected by gating on CD14+HLADR+cells.
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