Enzymatic synthesis of oligo-d-galactofuranosides and l-arabinofuranosides: from molecular dynamics to immunological assays

Abstract

D-Galactofuranosyl-containing conjugates are ubiquitous in many pathogenic microorganisms, but completely absent from mammals. As they may constitute interesting pharmacophores, recent works have been dedicated to their preparation. Besides well-reported chemical procedures, enzymatic approaches are still limited, mainly due to the lack of the corresponding biocatalysts. Based on the similarity between chemical structures, the arabinofuranosyl hydrolase Araf51 from Clostridium thermocellum was expected to recognize both the L-Araf motif and its D-Galf analogue. Molecular dynamics and STD-NMR were firstly used to confirm this hypothesis and increase our knowledge of the active site. Interestingly, this arabinofuranosidase was not only able to hydrolyze galactosyl derivatives, but was also really efficient in catalyzing oligomerisations using p-nitrophenyl furanosides as donors. The structures of the products obtained were determined using mass spectrometry and NMR. Amongst them, all the possible regioisomers of di-arabino and -galactofuranosides were synthesized, and the ratio of each regioisomer was easily tuned with respect to the reaction time. Especially, the galactofuranobioside displaying the biologically relevant sequence beta-D-Galf-(1,6)-beta-D-Galf was enzymatically prepared for the first time. All fractions going from di- to penta-arabino- and galactofuranosides were tested for their ability in eliciting the production of TNF-alpha. Interesting immunological properties were observed with arabinofuranosides as short as three sugar residues.

Exploiting sp2‐Hybridisation in the Development of Potent 1,5‐α‐l‐Arabinanase Inhibitors

“… A) GH93 arabinanases use a retaining catalytic mechanism involving a glycosyl–enzyme intermediate such that the reaction proceeds with net retention of stereochemistry (R=repeating 1,5‐α‐ l ‐arabinofuranosyl units). B) Structures of the known inhibitor of GH93 arabinanases 1 and of the corresponding hydroximolactones 2 and 3 , as well as the putative substrate 4 , relevant to the work described here.…”

“…Based on the putative conformational itinerary analysis that has been described, we felt that molecules bearing an sp 2 hybridised anomeric carbon such as in 2 and 3 might act as potent inhibitors of GH93 arabinanases. In addition, by utilising the chemistry employed for the synthesis of 2 and 3 , a more efficient preparation of the 4‐nitrophenyl glycoside 4 , prepared previously by chemoenzymatic and chemical methods, might yield a new substrate for measuring GH93 arabinanase activity directly. A more robust synthesis of 4 would make it more readily available and easier to use in further biochemical studies of these enzymes.…”

Chemo-enzymatic synthesis of an original arabinofuranosyl cluster: optimization of the enzymatic conditions

“…Determination of the kinetic parameter K m for the hydrolysis of pNP-Araf in standard conditions (0.2 mg/mL in 50 mM PBS pH 7, 1 mg/mL BSA) revealed a value of 0.15 mM (0.25 mM at 37 °C). 13 Optimum pH was determined for this reaction using 3 mM as the substrate concentrations and thus appeared to be comprised between 6.5 and 7.0. The enzyme proved to be very active on this artificial substrate revealing a k cat value of 415 min -1 and a specific activity of 2.85 10 3 min -1 mM -1 in accordance with previous studies.…”

“…In fact, due to the biological effect of previously published oligosaccharides, such glycoclusters are expected to exhibit improved immunostimulating properties. [13][14] However, the major drawback of our biocatalyzed procedure previously described, demonstrated to rely in the solubility of the thiophenol derivatives thus requiring the use of 50 equivalents. To overcome this problem, conditions of the enzymatic reaction were herein optimized through variations of the pH, the nature of the solvent and the concentration of the anti-oxidative agent dithiothreitol (DTT).…”

Functional roles of H98 and W99 and β2α2 loop dynamics in the α‐l‐arabinofuranosidase from Thermobacillus xylanilyticus

“…A previous STD-NMR study performed on CtAbf only revealed weak STD intensities for H-2, H-3 and H-4 of the l-Araf moiety bound in subsite )1 [27]. This is surprising and is in contradiction with a structural model of the Michaelis complex of CtAbf with A 3 X, which indicates that the l-Araf moiety was strongly stabilized in subsite )1 through multiple hydrogen bonds: OH-2 of the l-Araf moiety interacts both with the oxygen atom (2.6 Å ) of the nucleophile E292 (E298 in TxAbf) and with N172 (N175 in TxAbf), OH-3 interacts both with the side chain of E27 (E28 in TxAbf) and with the N72 backbone amide (instead of the C74 in TxAbf), while OH-5 interacts with the side chains of Y244 and Q352 (Y242 and Q347 in TxAbf, respectively).…”