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Abstract
Peptidoglycan induces pro-inflammatory responses which may lead to clinical symptoms of sepsis. To address this world-wide health problem, the signal transduction pathway responsible for these pro-inflammatory responses needs to be unraveled. This entails a proper understanding of structure-activity relationship of various part structures of peptidoglycan (PGN) with different receptors (CD14, TLR, NOD, PGRP) of the host cell. To unravel the complicated signaling involved, and identify various receptors involved in this pathway several peptidoglycan fragments were synthesized and biologically evaluated. PGN are large polymers composed of alternating (1-4)-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues, which are cross-linked by short peptide bridges. Lysine is commonly the third amino acid of the peptide moieties of PGN of Gram-positive bacteria while Gram-negative bacteria have a diaminopimelic acid (DAP) residue at this position. Although lysine-containing compounds can be conveniently synthesized by standard solution- or polymer supported approaches, the chemical synthesis of DAP containing fragments is hampered by difficulties in the obtaining of this unusual amino acid. We developed a method to synthesize DAP by a cross metathesis reaction between readily available allyl- and vinyl glycine followed by a reduction of the double bond of the resulting compound. Using the synthetic methodology developed a series of PGN fragments were synthesized and biologically evaluated. Cellular activation studies were performed to evaluate synergism exhibited by these PGN fragments. The role of Nod proteins, a pattern recognition receptors, in synergism was investigated and a SAR with different PGN fragments were performed. Direct binding studies with Peptodoglycan receptor protein (PGRP), another pattern recognition receptor called was also evaluated using surface plasmon resonance to illustrate selectivity of different kinds of PGRPs towards different structural variations of PGN. AcHNHOOOOOHOOOnHOAcHNNHOOOR1HN2OONHH NOR22NH3HNNHR3O1R = H or Cross-linking3R = OH or NH2 2R = H (Lys) Gram-positive PGN = COOH ( DAP) Gram-negative PGNGeneral structure of PGN