Synthesis and Conjugation of D-Glycero-β-D-Mannoheptose 1-Monophosphate

Abstract

Pathogen-associated molecular patterns (PAMPs) are molecular motifs of microbes that are not produced by the host. PAMPs are recognized by pattern recognition receptors that lead to the activation of a host immune response. As such, they are promising molecular scaffolds for use in immune modulating therapies; however, there are challenges associated with translating them to the clinic. D-glycero-β-D-mannoheptose 1-monophosphate (HMP) is an example of a PAMP with a carbohydrate scaffold that shows promise for use as an immune agonist. However, administering this sugar without chemical modifications requires cell membrane permeabilization due to its limited ability to enter cells. Finding a route to administer this PAMP without altering the cell membrane is a significant challenge and limits its testing in animal models and potential use as a drug. To address this, we proposed to conjugate D-glycero-β-D-mannoheptose 1-monophosphate to spherical nucleic acids, which are DNA-nanoparticle conjugates known to enter cells unaided. In this work, a large-scale synthesis of aminoethyl-D-glycero-β-D-mannoheptose 1-monophosphate was performed. The incorporation of an amine on this compound enabled its derivatization with an azide to form N3-PEG4-7-O-(amidoethyl)-D-glycero-β-D-manno-heptopyranose phosphate, which was conjugated to dibenzocyclooctyne (DBCO)-functionalized oligonucleotides synthesized by the Bujold group at McMaster University. These N3-PEG4-7-O-(amidoethyl)-D-glycero-β-D-manno-heptopyranose phosphate-functionalized oligonucleotides were then densely functionalized onto gold nanoparticles to form the desired spherical nucleic acids.