Figure 4.
Synthesis of SSO conjugates and their metabolism in mouse plasma. (A) Synthesis routes of MOE-PS SSO conjugates. The stearic acid conjugate ORNst was obtained in two steps through coupling of a 5′-amino C6-phosphoramidite linker, followed by reaction with stearic N-hydroxy succinimide ester (A); the peptide conjugate ORN84 and the cholesterol conjugate (ORNch) were generated in two steps using a 5′-caged maleimide modifier phosphoramidite and reaction with the cysteine-modified CD84-peptide (B) or thiocholesterol (C), respectively. (DCA: dichloroacetic acid; MMTr: monomethoxy-trityl). (B–D) ORN84, ORNch and ORNst were incubated in mouse plasma for 26 h at 37°C. Aliquots of plasma were removed periodically, cleaned and analysed by LC–MS. ORN184 underwent hydrolysis at the peptide and the succinimide linker (b). ORN1ch underwent hydrolysis-mediated ring opening (C). ORN1st was barely affected under the conditions (D). Red arrows indicate inferred sites of cleavage, based upon the masses of fragments in the LC–MS chromatagrams.