Influence of O6 in Mannosylations Using Benzylidene Protected Donors: Stereoelectronic or Conformational Effects?

The stereoselective synthesis of β-mannosides and the underlying reaction mechanism have been thoroughly studied, and especially the benzylidene-protected mannosides have gained a lot of attention since the corresponding mannosyl triflates often give excellent selectivity. The hypothesis for the enhanced stereoselectivity has been that the benzylidene locks the molecule in a less reactive conformation with the O6 trans to the ring oxygen (O5), which would stabilize the formed α-triflate and subsequent give β-selectivity. In this work, the hypothesis is challenged by using the carbon analogue (C7) of the benzylidene-protected mannosyl donor, which is investigated in terms of diastereoselectivity and reactivity and by low-temperature NMR. In terms of diastereoselectivity, the C-7-analogue behaves similarly to the benzylidene-protected donor, but its low-temperature NMR reveals the formation of several reactive intermediate. One of the intermediates was found to be the β-oxosulfonium ion. The reactivity of the donor was found to be in between that of the “torsional” disarmed and an armed donor

Automated Solid-Phase Synthesis of Hyaluronan Oligosaccharides

Well-defined fragments of hyaluronic acid (HA) have been obtained through a fully automated solid-phase oligosaccharide synthesis. Disaccharide building blocks, featuring a disarmed glucuronic acid donor moiety and a di-<i>tert</i>-butylsilylidene-protected glucosamine part, were used in the rapid and efficient assembly of HA fragments up to the pentadecamer level, equipped with a conjugation-ready anomeric allyl function

Apparent association constants (<i>K</i>_a) for <i>Pa</i>AlgX_27–474 and <i>Pa</i>AlgJ_79–379 for short polymannuronic oligosaccharides at 298 K and pH 7 determined by the direct ESI-MS assay.

<p>NB: No Binding.</p><p>*Ligand name as referenced in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004334#ppat.1004334-Walvoort2" target="_blank">[78]</a>.</p><p>Apparent association constants (<i>K</i>_a) for <i>Pa</i>AlgX_27–474 and <i>Pa</i>AlgJ_79–379 for short polymannuronic oligosaccharides at 298 K and pH 7 determined by the direct ESI-MS assay.</p

Specific activities of wild-type and catalytic triad mutants of <i>Pp</i>AlgJ_75–370 and <i>Pa</i>AlgJ_79–379.

<p>Specific activities were measured at an initial concentration of 3.0 mM ACC. WT and specific mutants for each AlgJ construct are labelled below the x-axis. Specific activity upon the y-axis was measured as µmol min⁻¹ mg⁻¹.</p

The AlgJ signature motifs.

<p>(A) Cartoon representation of <i>Pp</i>AlgJ_75–370 with residues from the signature motif represented as sticks. The AlgJ signature motif residues have been coloured according to the amount of alginate acetylation observed <i>in vivo</i> in each alanine variant relative to WT AlgJ. Yellow represents impairment of between 50–60% acetylation; orange represents strong impairment with only 6–25% acetylation observed; and red represents ablation or between 0–5% observed acetylation. Residues represented as grey sticks are proposed to be involved in interactions with the AlgJ signature motif residues but have not been characterized <i>in vivo</i>. (B) Selected residues from the box area in panel A that participate in or are proposed to be involved in the hydrogen bonding network and are coloured as described in (A). Hydrogen-bonds are depicted as green dashes with distance given in angstroms (Å). (C) Residues participating in hydrophobic and van der Waals interactions with W193 (shown in orange) are depicted in grey. Hydrophobic and van der Waals interactions less than 3.7 Å are depicted with blue dashes.</p

Structure and topology of <i>Pp</i>AlgJ_75–370.

<p>(A) Cartoon representation of <i>Pp</i>AlgJ_75–370 with secondary structural elements labelled (α: α-helix; β: β-strand; and t: 3₁₀ helix). Residues at discontinuous points in the structure due to poor observed electron density are labelled and coloured red. The N- and C-termini of the protein are labelled N and C, respectively, and the terminal residue is coloured red. (B) Topology model of the <i>Pp</i>AlgJ_75–370 structure with secondary structural elements and termini labelled as in panel (A).</p

ESI mass spectra to examine polymannuronic acid binding.

<p>ESI mass spectra acquired for aqueous ammonium acetate (50 mM) solutions of (A) <i>Pa</i>AlgX_27–474 (P) (4 µM) and 50 mM ManA₆); (B) <i>Pa</i>AlgX_27–474(P) (4 µM) and 50 mM ManA₁₂); (C) <i>Pa</i>AlgJ_79–379 (P) (10 µM) and 50 mM ManA₆); (D) <i>Pa</i>AlgJ_79–379 (P) (10 µM) and 50 mM ManA₁₂). A reference protein scFv (4 µM) was added into the solutions of AlgX, Lyz was added as P_ref to AlgJ solutions. Ion peaks corresponding to higher charge states of AlgJ are labelled as filled red circles. P_ref refers to the reference protein scFv.</p

AlgX_27–474 is an <i>O</i>-acetyltransferase.

<p>Mass spectra of the acetyltransferase reactions in the presence (red, line a) and absence (blue, line b) of <i>Pa</i>AlgX_27–474. Major peaks are labelled: ManA10, mannuronic acid decamer; ManA10+Na, mannuronic acid decamer plus sodium; ManA10+OAc, mannuronic acid decamer plus acetate; ManA10+Na+OAc, mannuronic acid decamer plus sodium and acetate; ManA10+2 OAc, mannuronic acid decamer plus two acetates. The observed molecular weights of each product are denoted under their respective labels. The addition of acetate adds 42.01 Da to the mass of the polymer. The data has been deconvoluted and centroided using Agilent MassHunter software and represents uncharged exact masses.</p

Summary of data collection and refinement statistics.

a<p>Values in parentheses correspond to highest resolution shell.</p>b<p><i>R</i>_merge = Σ Σ |I(k)−<i>|/Σ I(k) where I(k) and <i> represent the intensity values of the individual measurements and the corresponding mean values. The summation is over all unique measurements.</i></i></p><i><i>c<p><i>R</i>_work = Σ ∥<i>F</i>obs|−k|<i>F</i>calc∥/|<i>F</i>obs| where <i>F</i>obs and <i>F</i>calc are the observed and calculated structure factors, respectively. <i>R</i>_free is the sum extended over a subset of reflections (4.2%) excluded from all stages of the refinement.</p>d<p>As calculated using MOLPROBITY <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004334#ppat.1004334-Chen2" target="_blank">[82]</a>.</p>e<p>Maximum-Likelihood based coordinate error as determined by PHENIX <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004334#ppat.1004334-Adams1" target="_blank">[64]</a>.</p><p>Summary of data collection and refinement statistics.</p></i></i

Proposed model for the <i>O</i>-acetylation of alginate.

<p>AlgI receives the currently unidentified acetyl donor molecule on its cytoplasmic face and shuttles the acetate or acetyl donor molecule through the inner membrane, where it is transferred to either AlgJ and/or AlgF. The acetyl donor may or may not be transferred between AlgJ and AlgF. From its intermediate location, the acetyl donor molecule is transferred to AlgX, which subsequently <i>O</i>-acetylates the alginate polymer. Although absolutely required for alginate <i>O</i>-acetylation the function of AlgF is currently unknown. We have demonstrated that AlgJ has activity on acetylated substrates thereby suggesting it fulfills a more direct role in the O-acetylation process.</p