Synthesis and evaluation of fluorescent Pam3Cys peptide conjugates.
Chirally pure R- and S-epimers of TLR2 ligand Pam3CysSK4 were prepared and separately conjugated to an OVA model epitope, in which lysine was replaced by azidonorleucine. The azide function in the conjugate permitted labelling with different fluorophores by use of strain-promoted 3+2 cycloaddition. The R-epimer of the labelled conjugates induced TLR2-dependent DC maturation, while S-epimer proved to be inactive. Combining the lipophilicity of Pam3CysSK4 ligand with fluorophores influenced the solubility of the resulting conjugates in an unpredictable way and only the conjugates labelled with Cy-5 were suitable for confocal fluorescence microscopy experiments. It was shown that both epimers of the Cy-5 labelled lipopeptides were internalized equally well, indicating TLR2-independent cellular uptake. The presented results demonstrate the usefulness of strain-promoted azide-alkyne cycloaddition in the labelling of highly lipophilic lipopeptides without disturbing the in vitro activity of these conjugates with respect to activation of TLR-2.
Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, The Netherlands.
Geoffroy P P
GP
Gential
Department of Immunohematology and Blood Transfusion, Leiden University Medical Centre, The Netherlands.
Nataschja I
NI
Ho
Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, The Netherlands.
Fabrizio
F
Chiodo
Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, The Netherlands.
Nico
N
Meeuwenoord
Department of Immunohematology and Blood Transfusion, Leiden University Medical Centre, The Netherlands.
Ferry
F
Ossendorp
Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, The Netherlands.
Herman S
HS
Overkleeft
Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, The Netherlands.
Gijs A
GA
van der Marel
Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, The Netherlands.
Dmitri V
DV
Filippov
D002460:Cell Line; D003545:Cysteine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000493:pharmacokinetics; D003713:Dendritic Cells / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry / Q000493:pharmacokinetics; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis; D008024:Ligands; D008074:Lipoproteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000493:pharmacokinetics; D015394:Molecular Structure; D012995:Solubility; D013329:Structure-Activity Relationship; D051195:Toll-Like Receptor 2 / Q000378:metabolism
D005456:Fluorescent Dyes; D016209:Interleukin-8; D008024:Ligands; D008074:Lipoproteins; D051195:Toll-Like Receptor 2; C069172:2,3-bis(palmitoyloxy)-2-propyl-1-palmitoylcysteine; D003545:Cysteine
Fluorescent labeling; Lipopeptides; Strain promoted cycloaddition; Toll-like receptor 2