Peptidoglycan (PGN) is a component of bacterial cell wall consisting of glycans and peptide chains forming a three-dimensional mesh-like structure outside the plasma membrane. PGN has been known as a stimulating component of innate immune system. PGN activates sensor proteins, nucleotide-binding oligomerization domain protein 1 (Nod1) and 2 (Nod2), which belong to Nod-like receptor (NLR) family,1-3 one of the major pathogen-recognizing receptor (PRR) families. Peptidoglycan recognition proteins (PGRP) are the other important protein families that recognize PGN. In addition, various kinds of enzymes and lectins have been proven to recognize PGN. However, the comprehensive analysis of the substrate structures of recognizing proteins has been not really conducted, because of the lack of pure PGN fragments. Herein, we report the chemical synthesis of the PGN fragment library in order to analyze various ligand/protein interactions. The PGN-fragments microarray was also developed for the rapid and quantitative analysis of the interactions, leading to understanding of the defense system against infection of bacteria.
1. Synthesis of PGN fragments and their glycan sequence-dependent Nod2 activation
Our previous studies revealed that MDP (MurNAc-L-Ala-γ-D-isoGln) showed the most potent activity in Nod2 stimulation. We also found that the activity was decreased as the glycan chain length and peptide chain length increased by using synthesized tetrasaccharide and octasaccharide fragments that contain GlcNAc-MurNAc (GM) repeating units (Figure 1, 2a~2d).4-6 These fragments with GM units are considered to be produced by the lysozyme in host organisms which cleaves 1,4-β-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in PGN. On the other hand, PGN fragments having more than two MurNAc-GlcNAc (MG) repeating units, which are expected to be produced by bacterial N-acetylglucosaminidase and release to the environment, had not been synthesized. Thus, we synthesized the library of PGN fragments (Figure 1) to explore their biological functions, especially the Nod2 activation.7
1-1. Synthetic strategy of PGN fragments
The syntheses of disaccharide analogues were carried out via the intermediate 3 as illustrated in scheme 1. Introduction of appropriate peptides to the liberated carboxylic acid 6 and hydrogenation gave the disaccharide fragments 1a, 1c and 1e.
Tetrasaccharide 9 was then synthesized by using 3 as common synthetic intermediate for both glycosyl donor and the acceptor (Scheme 2). Disaccharide donors 7a and 7b were prepared via cleavage of the allyl glycoside and subsequent conversion to the imidate forms (7a: trichloroacetimidate, 7b: N-phenyltrifluoroacetimidate). For the preparation of the tetrasaccharide 9, trichloroacetimidate 7a was first used as the glycosyl donor to couple with the glycosyl acceptor 8. However, the glycosylation between 7a and 8 in the presence of TMSOTf gave the desired tetrasaccharide 9 only in 16% yield, accompanied with 62% yield of recovered 8. The low yield was probably due to the low reactivity of 4-OH group of the disaccharide acceptor 8 caused by steric hindrance of 3-O-lactyl moiety in muramic acid moiety. We then used the N-phenyltrifluoroacetimidate 7b as the glycosyl donor which has similar high reactivity but improved stability in comparison to the corresponding trichloroacetimidates.8Excess acceptor 8 was used (ratio of donor : acceptor was 1 : 1.5) in order to promote the reaction. The yield of glycosylation was dramatically improved to give tetrasaccharide 9 in 61% yield. The glycan backbone 10 was then coupled with peptides to give the corresponding protected intermediates. All benzyl and benzylidene groups
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