Synthesis and Immunological Evaluation of Mannosylated Desmuramyl Dipeptides Modified by Lipophilic Triazole Substituents

Abstract

Muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine, MDP) is the smallest peptidoglycan fragment able to trigger an immune response by activating the NOD2 receptor. Structural modification of MDP can lead to analogues with improved immunostimulating properties. The aim of this work was to prepare mannosylated desmuramyl peptides (ManDMP) containing lipophilic triazole substituents to study their immunomodulating activities in vivo. The adjuvant activity of the prepared compounds was evaluated in the mouse model using ovalbumin as an antigen and compared to the MDP and referent adjuvant ManDMPTAd. The obtained results confirm that the α-position of D-isoGln is the best position for the attachment of lipophilic substituents, especially adamantylethyl triazole. Compound 6c exhibited the strongest adjuvant activity, comparable to the MDP and better than referent ManDMPTAd.

Keywords: desmuramyl peptide; immunostimulating activity; mannose; triazole.

Figure 1

Structure of MDP, DMP and mannosylated adamantyl-triazole DMP derivative.

Scheme 1

Reagents and conditions: (a): (i) Boc₂O, TEA, 1,4-dioxane, H₂O, 0 °C to rt, 90 min; (ii) R-N₃, Na ascorbate (aq), CuSO₄ × 5 H₂O (aq), Ar, 35 °C, 24 h, yield: 97% (1a), 58% (1b), 53% (1c); (b) TFA, dry CH₂Cl₂, rt, 2–4 h; yield: 87% (2a), 96% (2b), 75% (2c).

Scheme 2

Reagents and conditions: (a) H₂, 10% Pd/C, MeOH, 34 psi, overnight, 95%; (b) 2a, EDC × HCl, HOBt, TEA, 1,4-dioxane/CH₂Cl₂, 0 °C to rt, 48 h, 71%; (c) TFA, dry CH₂Cl₂, rt, 2.5 h, 95%.

Scheme 3

Reagents and conditions: (a) 2b, HBTU, TEA, THF/dry CH₂Cl₂, Ar (g), rt, 24 h, 53% (3b); (b) (i) 2c, n-butyl chloroformate/N-methylmorpholine, THF/dry CH₂Cl₂, Ar (g), −10 °C to rt, 3 h; (ii) NaHCO₃ (aq), rt, 65% (3c); (c) TFA, dry CH₂Cl₂, rt, 4 h, yield: 82% (4b), 89% (4c).

Scheme 4

Reagents and conditions: (a) 4a–c, EDC × HCl, HOBt, TEA, 1,4-dioxane/CH₂Cl₂, 0 °C to rt, 48 h, yield: 60% (5a), 69% (5b), 57% (5c); (b) H₂, 10% Pd/C, 96% EtOH, 40 psi, rt, 24 h, yield: 60% (6a), 76% (6b), 67% (6c).

Figure 2

The effect of synthesized DMPs on the production of total anti-OVA IgG (a) and subtype of anti-OVA IgG1 (b) in BALB/c mice immunized with OVA as an antigen. Bar graphs represent the average values of individual mice from each group (n = 5). * p < 0.05, ** p < 0.01 and *** p < 0.001 denote statistical significance between indicated groups.

Figure 3

The effect of synthesized DMPs on the production of the anti-OVA IgG2a subtype (a) and IgG1/IgG2a ratio (b) in BALB/c mice immunized with OVA as an antigen. Bar graphs represent the average values of individual mice from each group (n = 5). * p < 0.05 denotes statistical significance between indicated groups.

Figure 4

Molecular docking of ManDMPTAd (A), 6b (B) and 6c (C) in the proposed binding pocket on the concave surface of the LRR domain of NOD2. The stick model of different colors shows selected ligands, and the key amino acid residues of the NOD2 receptor around compounds have been displayed in the light blue stick model. Hydrogen bonds are shown as dark gray dashed lines.

Figure 4

Molecular docking of ManDMPTAd (A), 6b (B) and 6c (C) in the proposed binding pocket on the concave surface of the LRR domain of NOD2. The stick model of different colors shows selected ligands, and the key amino acid residues of the NOD2 receptor around compounds have been displayed in the light blue stick model. Hydrogen bonds are shown as dark gray dashed lines.