Influence of Mannosylation on Immunostimulating Activity of Adamant-1-yl Tripeptide by Rosana Ribic ´* a ), Lidija Habjanec b ), Ruz ˇa Frkanec b ), Branka Vranes ˇic ´ b ), and Srd - anka Tomic ´ a ) a )Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb (phone: þ 385-1-4606402; fax: þ 385-1-4606401; e-mail: rribic@chem.pmf.hr) b ) Institute of Immunology, Rockfellerova 2, HR-10000 Zagreb The mannosylated derivative of adamant-1-yl tripeptide ( d-(Ad-1-yl)Gly-l-Ala-d-isoGln) was prepared to study the effects of mannosylation on adjuvant (immunostimulating) activity. Mannosylated adamant-1-yl tripeptide (Man-OCH 2 CH(Me)CO-d-(Ad-1-yl)Gly-l-Ala-d-isoGln) is a non-pyrogenic, H 2 O-soluble, and non-toxic compound. Adjuvant activity of mannosylated adamantyl tripeptide was tested in the mouse model with ovalbumin as an antigen and in comparison to the parent tripeptide and peptidoglycan monomer (PGM, b-d-GlcNAc-(1 ! 4)-d-MurNAc-l-Ala-d-isoGln-mesoDAP(eNH 2 )-d- Ala-d-Ala), a well-known effective adjuvant. The mannosylation of adamantyl tripeptide caused the amplification of its immunostimulating activity in such a way that it was comparable to that of PGM. Introduction. – Natural and synthetic peptidoglycans, as well as their fragments of different molecular mass exhibit remarkable biological activities. It was established that they, in particular, affect the immune system of mammalian hosts [1] [2]. Peptidoglycan fragments of well-defined structures, the best known of which are muramyl peptides, have been extensively studied as possible adjuvants for human and animal vaccines [3–5]. Muramyl dipeptide (MDP, N-acetylmuramyl-l-alanyl-d- isoglutamine) is known as the smallest synthetic adjuvant molecule capable of replacing whole Mycobacteria in Freund)s adjuvant [5] . MDP is the structural fragment of peptidoglycan monomer (PGM; 1; Fig. 1) used in this work, a disaccharide pentapeptide b-d-GlcNAc-(1 ! 4)-d-MurNAc-l-Ala-d-isoGln-mesoDAP(eNH 2 )-d- Ala-d-Ala, originating from Brevibacterium divaricatum. Several hundred chemically defined MDP analogs and derivatives were synthesized to modulate the properties of the parent molecule. Replacement of the N-acetylmur- amyl moiety with various acyl groups represents an important approach in the design of new immunologically active MDP analogs. Up to now, our research in the field of potential adjuvants was directed towards desmuramyl peptides, which contain adamantylglycine and mannosylated adamantylglycine moieties bound to the essential part of MDP, l-Ala-d-isoGln. Thus, diastereoisomers of adamant-1-yl tripeptides 2a and 2b [6], and adamant-2-yl tripeptides 3a and 3b [7] ( Fig. 1) were synthesized and showed to exhibit adjuvant (immunostimulating) activities in vivo, in a well-defined mouse model. Furthermore, mannosylated adamant-1-yl and adamant-2-yl tripeptides, 4 and 5 ( Fig. 1), respectively, were prepared as a mixture of diastereoisomers due to the d-l-d and l-l-d sequence in the tripeptide portion ( d,l-AdGly-l-Ala-d-isoGln) [8]. Preliminary investigations of their immunostimulating activity in vivo indicated CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 1373 # 2012 Verlag Helvetica Chimica Acta AG, Zürich