Synlett, Table of Contents

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

^aSchool 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

^bSchool of Biological Sciences, The University of Auckland, 3a Symonds Street, Auckland 1142, New Zealand

^cMaurice Wilkins Centre, The University of Auckland, 3a Symonds Street, Auckland 1142, New Zealand

,

Alicia J. Didsbury

^bSchool of Biological Sciences, The University of Auckland, 3a Symonds Street, Auckland 1142, New Zealand

,

Julie D. McIntosh

^bSchool of Biological Sciences, The University of Auckland, 3a Symonds Street, Auckland 1142, New Zealand

,

Kristina Burkert

^bSchool of Biological Sciences, The University of Auckland, 3a Symonds Street, Auckland 1142, New Zealand

,

Ho Yeung

^bSchool of Biological Sciences, The University of Auckland, 3a Symonds Street, Auckland 1142, New Zealand

,

Geoffrey M. Williams

^aSchool 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

^bSchool of Biological Sciences, The University of Auckland, 3a Symonds Street, Auckland 1142, New Zealand

^cMaurice Wilkins Centre, The University of Auckland, 3a Symonds Street, Auckland 1142, New Zealand

,

Margaret A. Brimble*

^aSchool of Chemical Sciences, The University of Auckland, 23 Symonds Street, Auckland 1142, New Zealand Fax: +64(9)3737422 Email: m.brimble@auckland.ac.nz

^cMaurice Wilkins Centre, The University of Auckland, 3a Symonds Street, Auckland 1142, New Zealand

› Author Affiliations

All articles of this category

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.

Key words

Key words

lipopeptides - CuAAC - synthetic lipids - adjuvant - click chemistry

References and Notes
1 Kawai T, Akira S. Immunity 2011; 34: 637
2 Coffman RL, Sher A, Seder RA. Immunity 2010; 33: 492
3 Feldmann M, Steinman L. Nature 2005; 435: 612
4 Huynh AS, Chung WJ, Cho HI, Moberg VE, Celis E, Morse DL, Vagner J. J. Med. Chem. 2012; 55: 9751
5 Purcell AW, Zeng W, Mifsud NA, Ely LK, Macdonald WA, Jackson DC. J. Pept. Sci. 2003; 9: 255
6 Huang HI, Wu PY, Teo CY, Chen MN, Chen YC, Silin D, Tao MH. Int. J. Cancer 2004; 108: 696
7 Eriksson EM. Y, Jackson DC. Curr. Protein Pept. Sci. 2007; 8: 412
8 Kang JY, Nan X, Jin MS, Youn SJ, Ryu YH, Mah S, Han SH, Lee H, Paik SG, Lee JO. Immunity 2009; 31: 873
9 Coffman RL, Sher A, Seder RA. Immunity 2010; 33: 492
10 Wu W, Li R, Malladi SS, Warshakoon HJ, Kimbrell MR, Amolins MW, David SA. J. Med. Chem. 2010; 53: 3198
11 Zeng W, Horrocks KJ, Robevska G, Wong CY, Azzopardi K, Tauschek M, Robins-Browne RM, Jackson DC. J. Biol. Chem. 2011; 286: 12944
12 Cheng C, Jain P, Bettahi I, Pal S, Tifrea D, de la Maza LM. Vaccine 2011; 29: 6641
13 Zeng W, Gauci S, Ghosh S, Walker J, Jackson DC. Vaccine 2005; 23: 4427
14 Cai H, Sun ZY, Huang ZH, Shi L, Zhao YF, Kunz H, Li YM. Chem. Eur. J. 2013; 1962
15 Zeng W, Horrocks KJ, Robevska G, Wong CY, Azzopardi K, Tauschek M, Robins-Browne RM, Jackson DC. J. Biol. Chem. 2011; 286: 12944
16 Cai H, Huang ZH, Shi L, Zhao Y.-F, Kunz H, Li YM. Chem. Eur. J. 2011; 17: 6396
17 Yeung H, Lee DJ, Williams GM, Harris PW. R, Dunbar RP, Brimble MA. Synlett 2012; 23: 1617
18 Salunke DB, Shukla NM, Yoo E, Crall BM, Balakrishna R, Malladi SS, David SA. J. Med. Chem. 2012; 55: 3353
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.
20 Metzger JW, Wiesmüller K.-H, Jung G. Int. J. Pept. Protein Res. 1991; 38: 345
21 Harris PW. R, Yang SH, Brimble MA. Tetrahedron Lett. 2011; 52: 6024
22 Han Y, Albericio F, Barany G. J. Org. Chem. 1997; 62: 4307
23 Kopycinski J, Osman M, Griffiths PD, Emery VC. J. Med. Virol. 2009; 82: 94