Synthesis of bifunctional lipid analogs

A 15 carbon-long FA containing a photo-activatable diazerine and clickable alkyne group, pacFA, was synthesized in three steps from commercially available educts, as described in (25) (see the supplemental information for further details). Next, pacFA was coupled to D-erythro-sphingosine using a combination of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) and hydroxybenzotriazole as condensing reagents, yielding the photo-activatable and clickable C15-ceramide analog, pacCer (85% overall yield). pacPC was synthesized starting from 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (Avanti Polar Lipids) and pacFA under the action of N,N-dicyclohexylcarbodiimide and 4-dimethylaminopyridine (DMAP) with satisfactory yield (39%). pacDAG was synthesized in three steps starting from 1-oleoyl-sn-glycerol (Santa Cruz Biotechnology). First, the primary HO-group was protected with the triphenylmethyl protecting group (trityl-chloride/pyridine; 92% overall yield). The glycerol obtained was coupled with the pacFA using EDCI/DMAP activation (58% overall yield). The final deprotection step was achieved using trifluoroacetic acid to generate pacDAG (28% overall yield). pacPE was synthesized in three steps starting from 1-oleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (Avanti Polar Lipids). First, the amino-group was protected with the tert-butoxycarbonyl protecting group (di-tert-butyl dicarbonate/triethylamine; 98% overall yield). The ethanolamine obtained was coupled with pacFA using EDCI/DMAP activation in a good yield (52%). The final deprotection step was achieved with trifluoroacetic acid to generate pacPE (35% overall yield). pacSM was synthesized starting from sphingosylphosphorylcholine (lyso-SM d18:1; Avanti Polar Lipids) and pacFA under the action of EDCI/hydroxybenzotriazole (78% overall yield). pacGlcCer was synthesized from 1-β-D-glucosylsphingosine (Matreya) and pacFA in the presence of triphenylphosphine and dithiopyridine, essentially as described by (34) (62% overall yield). The synthesis of C1-deoxy-pacCer, C3-deoxy-pacCer, and C3-deoxy-N-methyl-pacCer is described in the supplemental information. All synthetic compounds were purified by thin-layer chromatography to a high degree (purity >98%) and their structures were confirmed by 1H and 13C NMR and ESI MS.