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International audience; Cryptosporidiosis is a poorly controlled zoonosis caused by the intestinal infection by a protozoan parasite, Cryptosporidium parvum (Cp), presenting a high prevalence in ruminant farms (cattle, sheep, goats). Young animals with an immature intestinal immune system are particularly susceptible to this infection. In a neonatal mouse model, we previously demonstrated the key role of innate immunity and in particular of CD11c+CD103+CD11b- dendritic cell (DC) subset incontrolling the acute phase of Cp infection [1]. During infection, in response to chemokine production by infected epithelial cells, newly recruited CD11c+CD103+CD11b- DC produce IL12 and IFNγ contributing to the elimination process of the parasite. According to the current common classification of DC in different species (human, mouse, pig, sheep, and chicken), this subpopulation can be identified as the cDC1 subset of conventional DC. The aim of this project was to better characterize intestinal DC subpopulations in lamb and to determine their role during Cp infection. As in the mouse model, the parasite invades and multiplies mainly in the ileum of young lambs. However, a peculiarity of young ruminants is the presence of a large ileal Peyer’s patch (lymphoid tissue) that extends all along the ileum. We characterized in various compartments of the lamb small intestine (lymphoid and non-lymphoid) the various mononuclear phagocyte populations by performing a phenotypic and functional analysis by flow cytometry and transcriptomic methods respectively. We followed the evolution of the cell subsets according to the age of the animals and during Cp infection. This work represents the first fine description of mononuclear phagocyte cell subsets in the small intestine of young lambs at homeostasis and during the course of C. parvum infection.

The characterization of TDP-α-D-glucose dehydrogenase (AtmS8), TDP-α-D-glucuronic acid decarboxylase (AtmS9), and TDP-4-keto-α-D-xylose 2,3-dehydratase (AtmS14), involved in Actinomadura melliaura AT2433 aminodideoxypentose biosynthesis, is reported. This study provides the first biochemical evidence that both deoxypentose and deoxyhexose biosynthetic pathways share common strategies for sugar 2,3-dehydration/reduction and implicates the sugar nucleotide base specificity of AtmS14 as a potential mechanism for sugar nucleotide commitment to secondary metabolism. In addition, a re-evaluation of the AtmS9 homologue involved in calicheamicin aminodeoxypentose biosynthesis (CalS9) reveals that CalS9 catalyzes UDP-4-keto-α-D-xylose as the predominant product, rather than UDP-α-D-xylose as previously reported. Cumulatively, this work provides additional fundamental insights regarding the biosynthesis of novel pentoses attached to complex bacterial secondary metabolites.

A simple method for the study of sugar-nucleotide-dependent multienzyme cascades is highlighted where the use of selectively (13)C-labeled sugar nucleotides and inverse (13)C detection NMR offers fast, direct detection and quantification of reactants and products and circumvents the need for chromatographic separation. The utility of the method has been demonstrated by characterizing four previously uncharacterized sugar nucleotide biosynthetic enzymes involved in calicheamicin biosynthesis.

We described the integration of the general reversibility of glycosyltransferase-catalyzed reactions, artificial glycosyl donors, and a high throughput colorimetric screen to enable the engineering of glycosyltransferases for combinatorial sugar nucleotide synthesis. The best engineered catalyst from this study, the OleD Loki variant, contained the mutations P67T/I112P/T113M/S132F/A242I compared with the OleD wild-type sequence. Evaluated against the parental sequence OleD TDP16 variant used for screening, the OleD Loki variant displayed maximum improvements in k cat /K m of >400-fold and >15-fold for formation of NDP–glucoses and UDP–sugars, respectively. This OleD Loki variant also demonstrated efficient turnover with five variant NDP acceptors and six variant 2-chloro-4-nitrophenyl glycoside donors to produce 30 distinct NDP–sugars. This study highlights a convenient strategy to rapidly optimize glycosyltransferase catalysts for the synthesis of complex sugar nucleotides and the practical synthesis of a unique set of sugar nucleotides.

A two-step strategy for disaccharide modulation using vancomycin as a model is reported. The strategy relies upon a glycosyltransferase-catalyzed 'reverse' reaction to enable the facile attachment of an alkoxyamine-bearing sugar to the vancomycin core. Neoglycosylation of the corresponding aglycon led to a novel set of vancomycin 1,6-disaccharide variants. While the in vitro antibacterial properties of corresponding vancomycin 1,6-disaccharide analogs were equipotent to the parent antibiotic, the chemoenzymatic method presented is expected to be broadly applicable.

Human milk oligosaccharides (HMOs) are recognized as benefiting breast-fed infants in multiple ways. As a result, there is growing interest in the synthesis of HMOs mimicking their natural diversity. Most HMOs are fucosylated oligosaccharides. α-l-Fucosidases catalyze the hydrolysis of α-l-fucose from the non-reducing end of a glucan. They fall into the glycoside hydrolase GH29 and GH95 families. The GH29 family fucosidases display a classic retaining mechanism and are good candidates for transfucosidase activity. We recently demonstrated that the α-l-fucosidase from Thermotoga maritima (TmαFuc) from the GH29 family can be evolved into an efficient transfucosidase by directed evolution ( Osanjo et al. 2007). In this work, we developed semi-rational approaches to design an α-l-transfucosidase starting with the α-l-fucosidase from commensal bacteria Bifidobacterium longum subsp. infantis (BiAfcB, Blon_2336). Efficient fucosylation was obtained with enzyme mutants (L321P-BiAfcB and F34I/L321P-BiAfcB) enabling in vitro synthesis of lactodifucotetraose, lacto-N-fucopentaose II, lacto-N-fucopentaose III and lacto-N-difucohexaose I. The enzymes also generated more complex HMOs like fucosylated para-lacto-N-neohexaose (F-p-LNnH) and mono- or difucosylated lacto-N-neohexaose (F-LNnH-I, F-LNnH-II and DF-LNnH). It is worth noting that mutation at these two positions did not result in a strong decrease in the overall activity of the enzyme, which makes these variants interesting candidates for large-scale transfucosylation reactions. For the first time, this work provides an efficient enzymatic method to synthesize the majority of fucosylated HMOs.

A nanoporous membrane system with directed flow carrying reagents to sequentially attached enzymes to mimic nature’s enzyme complex system was demonstrated. Genetically modified glycosylation enzyme, OleD Loki variant, was immobilized onto nanometer-scale electrodes at the pore entrances/exits of anodic aluminum oxide membranes through His6-tag affinity binding. The enzyme activity was assessed in two reactions—a one-step “reverse” sugar nucleotide formation reaction (UDP-Glc) and a two-step sequential sugar nucleotide formation and sugar nucleotide-based glycosylation reaction. For the one-step reaction, enzyme specific activity of 6–20 min(–1) on membrane supports was seen to be comparable to solution enzyme specific activity of 10 min(–1). UDP-Glc production efficiencies as high as 98% were observed at a flow rate of 0.5 mL/min, at which the substrate residence time over the electrode length down pore entrances was matched to the enzyme activity rate. This flow geometry also prevented an unwanted secondary product hydrolysis reaction, as observed in the test homogeneous solution. Enzyme utilization increased by a factor of 280 compared to test homogeneous conditions due to the continuous flow of fresh substrate over the enzyme. To mimic enzyme complex systems, a two-step sequential reaction using OleD Loki enzyme was performed at membrane pore entrances then exits. After UDP-Glc formation at the entrance electrode, aglycon 4-methylumbelliferone was supplied at the exit face of the reactor, affording overall 80% glycosylation efficiency. The membrane platform showed the ability to be regenerated with purified enzyme as well as directly from expression crude, thus demonstrating a single-step immobilization and purification process.

Covering: up to the end of May 2011 Glyco(randomization/diversification) is a term that encompasses strategies to diversify a core drug scaffold via enzymatic glycosylation to provide sets of analogs wherein the sole diversity element is a carbohydrate. This review covers the influence of glycosylation upon various drug properties, the classes of glycosyl-conjugating enzymes amenable to glyco(randomization/diversification) schemes, approaches to the synthesis of required substrates and specific examples of glycorandomized libraries utilizing both wild-type and engineered enzymes.

Warfarin 1, a vitamin K antagonist (VKA), is a potent inhibitor of vitamin K epoxide reductase (VKOR) and acts by inhibiting the regeneration of reduced vitamin K, an essential cofactor for the clotting cascade.[1] Warfarin, and other members of the 4-hydroxy coumarin family, were first used as a rodenticides before evolving into the most widely prescribed oral drug class to treat thromboembolic events, including atrial fibrillation and deep-vein thrombosis.[2] While recent preclinical and clinical studies have implicated anticoagulants such as heparins in the reduction of tumor burden, metastatic potential and increasing overall patient survival,[3] the utility of VKAs in the context of anticancer therapy has been restricted to the treatment of cancer-associated venous thromboembolism.[4] Many 4-hydroxycoumarin analogs have been synthesized, culminating in derivatives with improved anti-VKOR potency and/or ADME properties.[5] Despite both the natural abundance of 4-hydroxy coumarin analogs and extensive synthetic and semi-synthetic structural diversity, to the best of our knowledge the only glycosylated metabolites of warfarin described in the literature are four (C6-, C8-, C4- and C4’-) glucuronides as standards for warfarin phase II metabolism studies.[6] Given the precedent of glycosylation to modulate drug potency, mechanism, solubility, and ADME properties,[7] we therefore set out to assess the potential impact of glycosylation on 1 activity. Herein we describe the synthesis of a library of 38 differentially-glycosylated warfarin neoglycosides. While this study revealed glycosylation to generally eliminate the native anti-VKOR activity of the parent, certain warfarin neoglycosides displayed surprising increases in anticancer cytotoxicity activity (nearly 2 orders of magnitude over the parent). Cumulatively, this study highlights the ability of glycosylation to fundamentally change the mechanism of a well-known pharmacophore and also provides a new practical method to prepare (R) and (S)-warfarin by the derivatization of racemic warfarin with a chiral auxiliary. Neoglycorandomization takes advantage of the reactivity of unprotected free reducing sugars toward a methoxyamine-appended aglycon and enables rapid access to differentially glycosylated variants of targeted small molecules or proteins.[8] [9] For rapid warfarin neoglycosylation, aglycon 3 was prepared according to Scheme 1 in two simple steps. Specifically, reaction of warfarin 1 with methoxyamine hydrochloride in pyridine resulted in the formation of 2 in a quantitative yield. The following reduction of the imine 2 using tert-butylamine.borane complex under acidic conditions resulted in the formation of the desired methoxyamine-appended aglycon 3. The chemoselective neoglycosylation reaction of 3 with diverse free reducing sugars in DMF/AcOH (3:1) at 50°C provided the corresponding warfarin neoglycosides 4–41 with yields ranging from 2 to 70% (average overall 41%).[10] All library members were purified by standard chromatography and LC-MS was subsequently used to assess purity and confirm identity.[10] Scheme 1 Synthesis of neoglycosides library. a) MeONH2, pyridine, MeOH, 99%; b) t-Bu-NH2.BH3, 10% aq. HCl, ethanol/dioxane (1:3), 86%; c) reducing sugar, DMF/AcOH (3:1). As a first pass assessment, the cytotoxicity of the library members was evaluated on eight human cancer cell lines representing a panel of carcinomas including lung, colorectal, breast, CNS and ovarian. In this study, the neoglycosides 4–41 were compared to warfarin 1, warfarin oxime 2 and warfarin neoaglycon 3 (Figure 1). From this assessment, 23 of the 38 library members displayed a better activity than warfarin 1 in at least one cancer cell line. The human colorectal HCT-15 cell line was most sensitive to warfarin neoglycosides with nine library members displaying an IC50 below 20 µm (compared to an IC50 of 543 µm for 1), whereas NCI-H460 (human lung adenocarcinoma) was highly resistant. Interestingly, pentosylation (32–41, with the exception of the d-arabinoside 33) led to drastic increases in anticancer activity and, in some cases cell line specificity, compared to the parent warfarin 1. Specifically, the neoglycosides 38–41 presented an elevated potency from 8–70 fold across 7 cell lines while the effectiveness of members 22–24, 34 and 35 was generally limited to HCT-15. Figure 1 Summary of IC50 data from the high-throughput cytotoxicity assay of 1–41 (reciprocal values displayed). Comparisons were performed against warfarin (1), warfarin oxime (2) and warfarin aglycon (3). A complete table of IC50 data and corresponding ... Although warfarin is prescribed as a racemate, (S)-warfarin exhibits 2–5 times more anticoagulant activity than the (R) enantiomer.[11] To determine the impact of the C9 stereocenter upon the novel anticancer activities of respective neoglycosides, we developed a highly efficient three step process to prepare (R) and (S)-warfarin 1. Several approaches for the synthesis of optically pure warfarin 1 have been reported in the literature and while asymmetric synthesis is among the most attractive strategies to date,[12] limited availability of organocatalysts and a dependence upon chiral chromatography continue to limit preparative scale approaches to either warfarin enantiomer. Our approach to prepare (R) and (S)-warfarin in enantiopure form was achieved by derivatization of the racemate 42 to diastereomeric ketals (S)-43 and (R)-43 (Scheme 2), which, as anticipated, were chromatographically separable. The reaction of warfarin 1 with (2R,3R)-2,3-butanediol was unsuccessful due to a facile intramolecular cyclization to give the corresponding mixed ketal.[12a, 13] Alternatively, methylation of warfarin 1 with trimethylsilyldiazomethane readily afforded 42 followed by the protection of the ketone to give the diastereoisomers 43. Subsequent demethylation and the deprotection of the ketone of (S)-43 and (R)-43, accomplished in a single step by a treatment with a mixture of TFA/H2O, provided the desired (S)-warfarin and (R)-warfarin, respectively in 65% overall yield.[14] Starting with pure (S)-warfarin and (R)-warfarin, the corresponding d-xylose and d-lyxose neoglycosides (based upon the most active carbohydrates elucidated in the racemic screen, Figure 1) were subsequently synthesized according to the Scheme 1. To our surprise, the activities of the (R)- or (S)-warfarin derived neoglycosides were indistinguishable from 39 or 41, respectively. Scheme 2 Synthesis of optically pure warfarin neoglycosides. a) Me3SiCHN2, CH2Cl2, 76%; b) (2R,3R)-2,3-butanediol, p-TSA, HC(OEt)3, CH2Cl2, 86%; c) TFA/H2O (4:1), 100%. d) MeONH2, pyridine, MeOH, 99%; e) t-Bu-NH2.BH3, 10% aq. HCl, ethanol/dioxane (1:3), 86%; f) ... The 4-hydroxy coumarins, including warfarin, inhibit vitamin K epoxide reductase complex 1 (VKORC1), which is involved in the vitamin K cycle. As a consequence, the suppression of VKORC1 activity compromises the γ-carboxylation of vitamin K dependant clotting factors II, VII, IX and X.[15][1] To subsequently evaluate how the structural modification of warfarin influences the anticoagulant potential of library members, a subset of representative neoglycosides were tested in a standard single dose VKORC1 assay (Figure 2).[1a] At a final concentration of 1 µm of warfarin 1, the remaining VKOR activity was 44% of the control (vehicle alone). While neoaglycon 3 displayed a similar anti-VKOR profile (albeit ~2-fold less potent), a 100-fold excess of each neoglycoside tested was required to approach the anti-VKOR activity of warfarin. Thus, the glycosylation of warfarin prohibits interaction with VKOR in a manner reminiscent to other biomodifications (e.g. P450-catalyzed hydroxylation in phase I metabolism),[2a] where an increase in hydrophilicity is anticipated to prohibit anticoagulant activity in vivo.[1a] Figure 2 Vitamin K epoxide reductase activity as a function of warfarin analog concentration (µM). In conclusion, neoglycosylation has enabled the study of the impact of glycodiversification upon the activity of warfarin. In this study, we demonstrate that the glycosylation of warfarin can dramatically modulate both cancer cell line cytotoxicity as well as the VKOR inhibition of warfarin culminating in specific analogs which display a 70-fold increase in anticancer activity and a corresponding 100-fold lower anti-VKOR activity. Whether this remarkably 7,000-fold change in ‘specificity’ reflects the potential to achieve a useful therapeutic window in vivo remains to be determined. An efficient derivatization of warfarin, having potential for gram scale synthesis of pure (R)-and (S)-warfarin, has also been developed as part of this study and revealed the warfarin C9 stereochemistry to have no influence upon the novel activities of corresponding neoglycosides. While this study clearly illustrates the appended sugar to be critical for altering both the mechanism of action and potency of the parent drug, determination of the precise anticancer mechanism of action of warfarin neoglycosides remains to be determined.

Glycosyltransferases (GTs) constitute a predominant enzyme superfamily responsible for the attachment of carbohydrate moieties to a wide array of acceptors that include nucleic acids, polysaccharides, proteins, lipids, carbohydrates and medicinally relevant secondary metabolites (1, 2). The majority of GTs are LeLoir (sugar nucleotide-dependent) enzymes and utilize nucleoside diphosphate sugars (NDP-sugars) as donors for glycosidic bond formation (Fig. 1a). Recent studies have revealed certain GT-catalyzed reactions from bacterial secondary metabolism to be reversible, presenting new GT-catalyzed methods for NDP-sugar synthesis as well as the GT-catalyzed exchange (Fig. 1b) or transfer (Fig. 1c) of sugars attached to both simple glycoside sugar donors(3, 4) and complex natural product glycosides including glycopeptides, enediynes(5), macrolides(6), macrolactams(7), (iso)flavonoids(8) and polyenes(9). Of the few examples which employed ‘activated’ sugar donors (e.g., glycosyl halides or aromatic glycosides) for GT-catalyzed transglycosylation(3, 4), only indirect evidence for the intermediacy of sugar nucleotides was provided. Among GT-catalyzed ‘reverse’ reactions where NDP-sugar formation has been confirmed, NDP-sugar formation was thermodynamically disfavored (i.e., with NDP-sugar as product, Keq < 1)(5, 10, 11) and typically required a 10- to 100-fold excess of NDP for sugar nucleotide production. In an effort to address the severe thermodynamic limitations of GT-catalyzed reactions run in reverse, herein we report the use of specific activated aromatic glycosides as donors in such reactions dramatically alters the equilibrium of GT-catalyzed reactions and thereby enables a variety of novel transformations (Fig. 1d) including: i) an unique platform for the efficient enzymatic syntheses of novel NDP-sugars, ii) a coupled GT-catalyzed platform for the differential glycosylation of small molecules (including natural products and synthetic drugs/targets), and iii) a colorimetric readout upon glycosyltransfer amenable to high throughput formats for glycodiversification and glycoengineering which can be coupled to nearly any downstream sugar nucleotide-utilizing enzyme. Fig. 1 Representative GT-catalyzed reactions. a) Classical GT-catalyzed transformation wherein the sugar, presented in the form of a sugar nucleotide donor, is conjugated to an acceptor target of interest to provide a thermodynamically-favored glycoside product. ...

Directed evolution is a valuable technique to improve enzyme activity in the absence of a priori structural knowledge, which can be typically enhanced via structure-guided strategies. In this study, a combination of both whole-gene error-prone polymerase chain reaction and site-saturation mutagenesis enabled the rapid identification of mutations that improved RmlA activity toward non-native substrates. These mutations have been shown to improve activities over 10-fold for several targeted substrates, including non-native pyrimidine- and purine-based NTPs as well as non-native d- and l-sugars (both α- and β-isomers). This study highlights the first broadly applicable high throughput sugar-1-phosphate nucleotidyltransferase screen and the first proof of concept for the directed evolution of this enzyme class toward the identification of uniquely permissive RmlA variants.

International audience; Cryptosporidiosis is a poorly controlled zoonosis caused by an intestinal parasite, Cryptosporidium parvum (Cp), with a high prevalence in livestock (cattle, sheep, goats). Young animals are particularly susceptible to this infection due to the immaturity of their intestinal immune system. In a neonatal mouse model, we previously demonstrated the importance of the innate immunity and in particular of CD11c+CD103+CD11b-conventional dendritic cells (cDC) among mononuclear phagocytes (MP) in controlling the acute phase of Cp infection. During infection, in response to chemokine production by infected epithelial cells, newly recruited cDCs produce IL12 and IFNγ contributing to the elimination of the parasite. According to the well-established mouse cDC classification, this Batf3+DC subpopulation corresponds to the cDC1 subset. The aim of this project was to better characterize intestinal MP subpopulations in neonatal lamb and calf at homeostasis and during Cp infection. As in the mouse model, the parasite invades and multiplies mainly in the ileum of animals. However, a peculiarity of young ruminants is the presence of a large ileal Peyer’s patch (lymphoid tissue) that extends all along the ileum. MP were therefore analyzed in lymphoid and non-lymphoid intestinal tissues of lambs and claves. We performed phenotypic and functional analyses of mononuclear phagocytes by flow cytometry and by transcriptomic methods (FLUIDIGM®) respectively, in the distal jejunum, jejunal and ileal Peyer’s patches. We characterized a population of macrophages and three subpopulations of cDC. We demonstrated that the subset identified as cDC1, according to the current common classification of cDC in different species (human, mouse, pig, sheep and chicken), increases with the age of animal. This might be linked with the decrease in sensitivity to C. parvum observed with the age. We are currently investigating the evolution of cDC1 subset during C. parvum infection.