Synthesis of novel adamantylacetyl derivative of peptidoglycan monomer--biological evaluation of immunomodulatory peptidoglycan monomer and respective derivatives with lipophilic substituents on amino group.

Novel synthetic analogue of immunomodulatory peptidoglycan monomer 1 (PGM), (adamant-1-yl)-CH2CO-PGM (2), was prepared by acylation of epsilon-amino group of diaminopimelic acid with symmetrical (adamant-1-yl)-acetic acid anhydride in the presence of triethylamine. The product was isolated by gel filtration on Sephadex G-25, followed by ion exchange chromatography on SP-Sephadex C-25. The susceptibility of (adamant-1-yl)-CH2CO-PGM to hydrolysis with N-acetylmuramyl-L-alanine amidase was demonstrated, and the product of hydrolysis, (adamant-1-yl) CH2CO-pentapeptide 3, was characterized. Both 2 and 3 are water soluble and non-pyrogenic compounds. Immunomodulatory activity of PGM (adamant-1-yl)-CH2CO-PGM and structurally related derivative Boc-Tyr-PGM was compared in experiments in vivo, in mice, using ovalbumin (OVA) as an antigen. All three tested compounds exhibited comparable immunostimulating effects with respect to the induction of anti-ovalbumin immunoglobulin G. The results of evaluation of biological activity show that the substitution of free amino group in the parent peptidoglycan molecule with bulky lipophilic substituents did not affect the susceptibility to hydrolysis with N-acetylmuramyl-L-alanine amidase and did not alter markedly the immunostimulating activity. The results also indicate that the free amino group in the peptide chain is not a necessary requirement in the mechanism of immunostimulation of tested immunomodulators.

Comparative study of the effects of peptidoglycan monomer and structurally related adamantyltripeptides on humoral immune response to ovalbumin in the mouse.

Peptidoglycan monomer, GlcNAc-MurNAc-L-Ala-D-isoGln-mesoDAP(omega NH2)-D-Ala-D-Ala (PGM) originating from Brevibacterium divaricatum and synthetic adamantyltripeptides, diastereoisomers of D,L-(adamant-2-yl)-Gly-L-Ala-D-isoGln (AdTP1 and AdTP2) exhibit immunomodulating activity. An experimental model in the mouse has been established with suboptimal amounts of ovalbumin (OVA) as the immunogen, and parallel testing of adjuvant activity of these three immunomodulators was carried out in Balb/c, C57B16 or CBA mice. Tested compounds (100 or 200 micrograms/mouse) mixed with OVA in saline (50 micrograms/mouse) were administered s.c. Anti-OVA was assayed by ELISA in the sera of mice taken 7 days after the boosters (given on days 14 and 28). The treatment with PGM and one of the diastereoisomers, AdTP2, resulted in significantly higher increase in anti-OVA IgG levels (stimulation index up to 46) with respect to controls and groups treated with AdTP1. The effect of AdTP2 treatment was comparable to that of PGM in most experiments after the first booster, but after the second booster PGM exhibited markedly better effect. PGM and AdTP2 also induced markedly higher levels of IgG1 and IgG2 anti-OVA subclasses than detected in controls and AdTP1 treated mice, indicating that these two immunomodulators might upregulate both Th1-like and Th2-like immune responses.