Kilogram scale chemical synthesis of 2'-fucosyllactose.

A scalable synthetic procedure to high quality 2'-fucosyllactose, the most abundant oligosaccharide in human breast milk, has been designed and validated in kilogram scale. The synthetic route has been developed to suit industrial environment and contains only a single chromatographic purification step.

Production of HMOs using microbial hosts - from cell engineering to large scale production.

Human Milk Oligosaccharides (HMOs) constitute an important, highly abundant part of mothers' milk delivering many health benefits to the neonate. Until recently, limited availability of HMOs has prevented their use in infant nutrition and impeded research into their biological effects. The shift from chemical synthesis to biotechnological manufacturing has made them accessible in quantities and at prices that are within reach for commercial applications, including infant formula. It accelerated the studies in the field of pre-clinical and clinical HMO biology. This review gives a short overview of HMO manufacturing from the design and optimization of the microbial cell factory and the production of HMOs in the industrial fermentation process to the purification in the downstream process necessary to obtain a final product. Moreover, the transition from chemistry to biotechnology and the current regulatory landscape and commercialization progress are briefly reviewed.

Design of an α-L-transfucosidase for the synthesis of fucosylated HMOs.

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.

Novel efficient routes to indoxyl glycosides for monitoring glycosidase activities.

A new efficient synthesis for broad access to indoxyl glycosides was developed. Indoxylic acid allyl ester linked to a sugar structure served as the key intermediate in this route. Selective ester cleavage and mild decarboxylation led to the corresponding indoxyl glycosides in good yields. This synthesis was applied for preparation of indoxyl glycosides of fucose, sialic acid, and 6'-sialyl lactose.

Resistance of galactoside-terminated alkanethiol self-assembled monolayers to marine fouling organisms.

Self-assembled monolayers (SAMs) of galactoside-terminated alkanethiols have protein-resistance properties which can be tuned via the degree of methylation [Langmuir 2005, 21, 2971-2980]. Specifically, a partially methylated compound was more resistant to nonspecific protein adsorption than the hydroxylated or fully methylated counterparts. We investigate whether this also holds true for resistance to the attachment and adhesion of a range of marine species, in order to clarify to what extent resistance to protein adsorption correlates with the more complex adhesion of fouling organisms. The partially methylated galactoside-terminated SAM was further compared to a mixed monolayer of ω-substituted methyl- and hydroxyl-terminated alkanethiols with wetting properties and surface ratio of hydroxyl to methyl groups matching that of the galactoside. The settlement (initial attachment) and adhesion strength of four model marine fouling organisms were investigated, representing both micro- and macrofoulers; two bacteria (Cobetia marina and Marinobacter hydrocarbonoclasticus), barnacle cypris larvae (Balanus amphitrite), and algal zoospores (Ulva linza). The minimum in protein adsorption onto the partially methylated galactoside surface was partly reproduced in the marine fouling assays, providing some support for a relationship between protein resistance and adhesion of marine fouling organisms. The mixed alkanethiol SAM, which was matched in wettability to the partially methylated galactoside SAM, consistently showed higher settlement (initial attachment) of test organisms than the galactoside, implying that both wettability and surface chemistry are insufficient to explain differences in fouling resistance. We suggest that differences in the structure of interfacial water may explain the variation in adhesion to these SAMs.

Synthesis of the Trypanosoma cruzi LPPG heptasaccharyl myo-inositol.

Synthesis of the heptasaccharyl myo-inositol found in Trypanosoma cruzi lipopeptidophosphoglycan was accomplished using a convergent assembly of three building blocks. The target compound is the first complete 2-aminoethyl phosphonic acid substituted glycan related to the glycosylphosphatidylinositol anchor family to be synthesized. The order of assembly enables synthesis of phosphoinositol oligosaccharides related to other glycosylinositolphospholipids in Tr. cruzi, the protozoan parasite causing Chagas' disease, which is endemic in South America.

Synthesis of the core tetrasaccharide of Trypanosoma cruzi glycoinositolphospholipids: Manp(alpha1-->6)-Manp(alpha1-->4)-6-(2-aminoethylphosphonic acid)-GlcNp(alpha1-->6)-myo-Ins-1-PO4.

[structure: see text] Synthesis of the core tetrasaccharide Manp(alpha1-->6)-Manp(alpha1-->4)-6-(2-aminoethylphosphonic acid)-GlcNp(alpha1-->6)-myo-Ins-1-PO4, found in glycoinositolphospholipids of Trypanosoma cruzi parasites, is described. The key building block, 6-O-(2-azido-3-O-benzyl-6-O-((2-benzyloxycarbonylaminoethyl)phosphonic acid benzyl ester)-2-deoxy-alpha-D-glucopyranosyl)-1-di-O-benzylphosphoryl-4,5-O-isopropylidene-2,3-O-(D-1,7,7-trimethyl[2,2,1]bicyclohept-6-ylidene)-D-myo-inositol, was synthesized using a partially protected glucosyl D-camphorinositolphosphate and a (2-benzyloxycarbonylaminoethyl)phosphonic acid derivative in a regioselective phosphonate esterfication. Elongation with ethyl 2-O-benzoyl-3,4,6-tri-O-benzyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzyl-1-alpha-D-thiomannopyranoside using dimethyl(methylthio)sulfonium trifluoromethanesulfonate gave a fully protected tetrasaccharide which was successfully deprotected subsequently with sodium methoxide, sodium in liquid ammonia, and aq hydrochloric acid to give title compound.

Synthesis and self-assembly of galactose-terminated alkanethiols and their ability to resist proteins.

The synthesis of two galactose-terminated alkanethiols with the structural formula X-OC2H5NHCO(CH2)15SH (X = 2,3,4,6-tetra-O-methyl-beta-D-Gal or beta-D-Gal) is described. Single-component and mixed self-assembled monolayers (SAMs) of the methylated and nonmethylated compounds were prepared on gold and subsequently characterized with ellipsometry, contact angle goniometry, and infrared reflection-absorption spectroscopy. Studies of the irreversible protein adsorption onto the SAMs using ex-situ ellipsometry revealed very low levels of fibrinogen and lysozyme adsorption onto mixed SAMs displaying advancing water contact angles between 24 degrees and 45 degrees and below 45 degrees , respectively. A monomethylated compound (X = 6-O-methyl-beta-D-Gal) was also synthesized and assembled on gold. This particular compound was found to possess wettability properties corresponding to the low adsorption regime of the mixed SAMs, and the results from the same set of fibrinogen and lysozyme adsorption experiments showed very low levels of protein adsorption. Our findings suggest that the protein rejecting properties rely on a fine balance between the surface energy and/or hydrogen bond donating/accepting properties of the SAM surface.

Mimicking the properties of antifreeze glycoproteins: synthesis and characterization of a model system for ice nucleation and antifreeze studies.

Synthesis of beta-D-Gal-(1 --> 3)-beta-D-GalNAc coupled to HOC2H4NHCOC15H30SH is described. This compound was coadsorbed at various proportions with C2H5OC2H4NHCOC15H30SH to form statistically mixed self-assembled monolayers (SAMs) on gold in an attempt to mimic the properties of the active domain in antifreeze glycoproteins (AFGPs). The monolayers were characterized by null ellipsometry, contact angle goniometry, X-ray photoelectron spectroscopy, and infrared reflection-absorption spectroscopy. The disaccharide compound adsorbed preferentially, and SAMs prepared at a solution molar ratio >0.3 displayed total wetting. The mixed SAMs showed well-organized alkyl chains up to a disaccharide surface fraction of 0.8. The amount of gauche conformers in the alkyls increased rapidly above this point, and the monolayers became disordered and less densely packed. Furthermore, the generated mixed SAMs were subjected to water vapor at constant relative humidity and the subsequent ice crystallization on a cooled substrate was monitored via an optical microscope. Interestingly, rapid crystallization occurred within a narrow range of temperatures on mixed SAMs with a high disaccharide content, surface fraction >0.3. The reported crystallization temperatures and the ice layer topography were compared with results obtained for a much simpler reference system composed of -OH/-CH3 terminated n-alkanethiols in order to account for changes in topography of the water/ice layer with surface energy. Although preliminary, the obtained results can be useful in the search for the molecular mechanism behind the antifreeze activity of AFGPs.