Convergent Chemoenzymatic Strategy to Deliver a Diversity of Shigella flexneri Serotype-Specific O-Antigen Segments from a Unique Lightly Protected Tetrasaccharide Core

Abstract

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Redirecting substrate regioselectivity using engineered ΔN123-GBD-CD2 branching sucrases for the production of pentasaccharide repeating units of S. flexneri 3a, 4a and 4b haptens

“…With the exception of mutant W2135F-F2136I (the least active mutant on sucrose alone at pH 4.7), all enzymes were able to glucosylate tetrasaccharide ABC′D′ (present at 50 mM) and 1 M of sucrose. LC/MS analysis 8 revealed that the two previously reported monoglucosylated tetrasaccharides P1 and P2 obtained with the parental enzyme (retention times 21.65 min and 21.9 min, respectively, and mass values m/z: 1057.23596 and 1057.23564, respectively) were produced in varying amounts depending on the enzyme (Figs. 1, 2).…”

“…The library of 22 variants, generated from ΔN 123 -GBD-CD2 branching sucrase 6 and comprising enzymes previously shown to exhibit a good ability to recognize bulky acceptor substrates, was screened for the glucosylation of tetrasaccharide ABC′D′. To limit the quantity of tetrasaccharide used, the reaction volume was scaled down to 10 µL and the reaction pH was fixed at 4.7 to avoid degradation of the chloroacetyl protecting group on C′ unit 8 . For all enzymes, the activity values on sucrose alone were lower at pH 4.7 than at pH 5.75 (the optimal pH of the parental enzyme 13,14 ) but remained sufficiently high to allow screening (data not shown).…”

“…In the continuity of these studies, we recently tested the ability of branching sucrases-native GH70 sucrose-active transglucosylases specialized in dextran branching-to regioselectively glucosylate a lightly protected tetrasaccharide (allyl α-l-rhamnopyranosyl-(1 → 2)-α-l-rhamnopyranosyl-(1 → 3)-(2-O-chloroacetylα-l-rhamnopyranosyl)-(1 → 3)-2-deoxy-2-trichloroacetamido-β-d-glucopyranoside, named herein ABC′D′), which was designed and chemically synthesized to serve as a common scaffold to precursors of Shigella flexneri serotype-specific haptens that could enter in the composition of a broad serotype coverage Shigella vaccine, while respecting criteria imposed by the enzyme plasticity. Indeed, the regioselective enzymatic α-d-glucosylation of ABC′D′ led to several pentasaccharides, some of which featuring the glucosylation pattern of Shigella flexneri O-antigens 8 . While conversion was low, this promising achievement was the proof-of-concept that cutting-edge combination of a fine-tuned chemically synthesized ABC′D′ tetrasaccharide scaffold and sucrose-active enzymes could be a way forward to a multivalent synthetic glycan-based vaccines.…”

Exploratory N‐Protecting Group Manipulation for the Total Synthesis of Zwitterionic Shigella sonnei Oligosaccharides

“…[39] Subsequent propenyl hydrolysis promoted by iodine in the presence of sodium bicarbonate [31] furnishedh emiacetal 14 in an acceptable yield. Replacing I 2 / NaHCO 3 by N-iodosuccinimide [40] (NIS) enabled as mootherh ydrolysis step in the case of the Fmoc analogue to furnishhemiacetal 15 in af airly improved yield. Conventional reactiono f the former with (N-phenyl)trifluoroacetimidoyl chloride [41] in the presence of cesium carbonate gave the (N-phenyl)trifluoroacetimidate donor 16.T MSOTf-mediated glycosylationo ft he latter with the knownA AT acceptor 8 at À30 8Cp roceeded in high yield (95 %) albeit furnishing a % 5:1m ix of two glycosylation products in addition to the elimination product (15 %) (Scheme S2, Entry 1).…”

“…Subsequent propenyl hydrolysis promoted by iodine in the presence of sodium bicarbonate [31] furnished hemiacetal 14 in an acceptable yield. Replacing I 2 /NaHCO 3 by N ‐iodosuccinimide [40] (NIS) enabled a smoother hydrolysis step in the case of the Fmoc analogue to furnish hemiacetal 15 in a fairly improved yield. Conventional reaction of the former with ( N ‐phenyl)trifluoroacetimidoyl chloride [41] in the presence of cesium carbonate gave the ( N ‐phenyl)trifluoroacetimidate donor 16 .…”