Synthesis of muramyl dipeptide analogues as immunomodulatory agents

Abstract

Peptidoglycan occurs naturally in cell wall structures of almost all types of bacteria. Muramyl dipeptide (MDP) is a fragment of peptidoglycan, which consists of N-acetylmuramic acid that is attached to a dipeptide of L-alanine and D-iso-glutamine. Peptidoglycan is a potent immunostimulant and MDP has been identified as the minimal structure required of peptidoglycan for activating the innate immune system. MDP engages the nucleotide-binding oligomerization domain-containing protein 2 (NOD2) receptor and activates the receptor. Importantly, MDP shows a promising vaccine adjuvant property. However, high toxicity, hydrophilicity, rapid elimination from the biological system, and other drawbacks associated with MDP prevent it from being used in clinics. In this study, novel MDP analogues have been designed and synthesized in order to improve the molecule’s biological activity. The modification in these analogues was applied within the dipeptide component, that is, using an artificial aromatic amino acid residue in place of the D-iso-glutamine residue in natural MDP. N-acetylation of 4-amino-3-nitrobenzoic acid was the starting point of the synthesis pathway. Next, the nitro group was reduced and coupled with L-alanine to form the main building block 14. The incorporation of different lengths of lipid chains at the C-terminal of the dipeptide mimic 14 resulted in lipophilic dipeptide analogues 11, 12 and 13. The dipeptide mimics 11 - 14 were then coupled with an N-acetylmuramic acid derivative 36 to provide protected MDP analogues 37 - 40. The first set of MDP analogues 4 - 6 were obtained by applying 37, 38, and 39 under the global deprotection reaction using Pd/C as the catalyst in a hydrogen atmosphere to remove both benzyl and benzylidene groups. The second set of analogues 7 - 9 were formed by treating 37 - 39 with trifluoroacetic acid to remove only the benzylidene group. Attempts at forming hydrophilic mimics 2 and 3 were not entirely successful due to a technical obstacle during the last step of the deprotection reactions. Ultimately, biological evaluation will determine the potential of these analogues to act as ligands for NOD2 and their immune-modulatory/stimulatory property.