- Reversible derivatization of sugars with carbobenzyloxy groups and use of the derivatives in solution-phase enzymatic oligosaccharide synthesis
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Simple protocols for attaching and detaching carbobenzyloxy (Cbz) groups at the reducing end of sugars was developed. Briefly, lactose was converted into its glycosylamine, which was then acylated with carbobenzyloxy chloride in high overall yield. The obtained lactose Cbz derivative was used in sequential glycosylations using glycosyltransferases and nucleotide sugars in aqueous buffers. Isolation of the reaction products after each step was by simple C-18 solid-phase extraction. The Cbz group was removed by catalytic hydrogenolysis or catalytic transfer hydrogenation followed by in situ glycosylamine hydrolysis. In this way, a trisaccharide (GlcNAc-lactose), a human milk tetrasaccharide (LNnT), and a human milk pentasaccharide (LNFPIII) were prepared in a simple and efficient way.
- Norberg, Thomas,Kallin, Elisabet,Blixt, Ola
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- Novel Reversible Fluorescent Glycan Linker for Functional Glycomics
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To aid in generating complex and diverse natural glycan libraries for functional glycomics, more efficient and reliable methods are needed to derivatize glycans. Here we present our development of a reversible, cleavable bifunctional linker 3-(methoxyamino)propylamine (MAPA). As the fluorenylmethyloxycarbonate (Fmoc) version (F-MAPA), it is highly fluorescent and efficiently derivatizes free reducing glycans to generate closed-ring derivatives that preserve the structural integrity of glycans. A library of glycans were derivatized and used to generate a covalent glycan microarray using N-hydroxysuccinimide derivatization. The array was successfully interrogated by a variety of lectins and antibodies, demonstrating the importance of closed-ring chemistry. The glycan derivatization was also performed at large scale using milligram quantities of glycans and excess F-MAPA, and the reaction system was successfully recycled up to five times, without an apparent decrease in conjugation efficiency. The MAPA-glycan is also easy to link to protein to generate neoglycoproteins with equivalent glycan densities. Importantly, the MAPA linker can be reversibly cleaved to regenerate free reducing glycans for detailed structural analysis (catch-and-release), often critical for functional studies of undefined glycans from natural sources. The high conjugation efficiency, bright fluorescence, and reversible cleavage of the linker enable access to natural glycans for functional glycomics.
- Wei, Mohui,McKitrick, Tanya R.,Mehta, Akul Y.,Gao, Chao,Jia, Nan,McQuillan, Alyssa M.,Heimburg-Molinaro, Jamie,Sun, Lijun,Cummings, Richard D.
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p. 2897 - 2908
(2019/11/11)
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- The chemical synthesis of human milk oligosaccharides: Lacto-N-neotetraose (Galβ1→4GlcNAcβ1→3Galβ1→4Glc)
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The discovery of innovative methods that offer new capabilities for obtaining individual oligosaccharides from human milk will help to improve understanding their roles and boost practical applications. The total chemical synthesis of lacto-N-neotetraose
- Bandara, Mithila D.,Stine, Keith J.,Demchenko, Alexei V.
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- Biochemical characterization of Helicobacter pylori α1–3-fucosyltransferase and its application in the synthesis of fucosylated human milk oligosaccharides
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Fucosylated human milk oligosaccharides (HMOs) have important biological functions. Enzymatic synthesis of such compounds requires robust fucosyltransferases. A C-terminal 66-amino acid truncated version of Helicobacter pylori α1–3-fucosyltransferase (Hp3FT) is a good candidate. Hp3FT was biochemically characterized to identify optimal conditions for enzymatic synthesis of fucosides. While N-acetyllactosamine (LacNAc) and lactose were both suitable acceptors, the former is preferred. At a low guanosine 5′-diphospho-β-L-fucose (GDP-Fuc) to acceptor ratio, Hp3FT selectively fucosylated LacNAc. Based on these enzymatic characteristics, diverse fucosylated HMOs, including 3-fucosyllactose (3-FL), lacto-N-fucopentaose (LNFP) III, lacto-N-neofucopentaose (LNnFP) V, lacto-N-neodifucohexaose (LNnDFH) II, difuco- and trifuco-para-lacto-N-neohexaose (DF-paraLNnH and TF-para-LNnH), were synthesized enzymatically by varying the ratio of the donor and acceptor as well as controlling the order of multiple glycosyltransferase-catalyzed reactions.
- Bai, Jing,Wu, Zhigang,Sugiarto, Go,Gadi, Madhusudhan Reddy,Yu, Hai,Li, Yanhong,Xiao, Cong,Ngo, Alice,Zhao, Baohua,Chen, Xi,Guan, Wanyi
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- Sequential One-Pot Multienzyme Chemoenzymatic Synthesis of Glycosphingolipid Glycans
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Glycosphingolipids are a diverse family of biologically important glycolipids. In addition to variations on the lipid component, more than 300 glycosphingolipid glycans have been characterized. These glycans are directly involved in various molecular recognition events. Several naturally occurring sialic acid forms have been found in sialic acid-containing glycosphingolipids, namely gangliosides. However, ganglioside glycans containing less common sialic acid forms are currently not available. Herein, highly effective one-pot multienzyme (OPME) systems are used in sequential for high-yield and cost-effective production of glycosphingolipid glycans, including those containing different sialic acid forms such as N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid (Kdn), and 8-O-methyl-N-acetylneuraminic acid (Neu5Ac8OMe). A library of 64 structurally distinct glycosphingolipid glycans belonging to ganglio-series, lacto-/neolacto-series, and globo-/isoglobo-series glycosphingolipid glycans is constructed. These glycans are essential standards and invaluable probes for bioassays and biomedical studies.
- Yu, Hai,Li, Yanhong,Zeng, Jie,Thon, Vireak,Nguyen, Dung M.,Ly, Thao,Kuang, Hui Yu,Ngo, Alice,Chen, Xi
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p. 10809 - 10824
(2016/11/29)
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- Method for preparation of the tetrasaccharide lacto-N-neotetraose (LNnt) containing N-acetyllactosamine
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The present invention relates to a method for preparation of the tetrasaccharide lacto-N-neotetraose (LNnt, formula (I)) especially in large scale, as well as intermediates in the synthesis, a new crystal form (polymorph) of LNnt, and the use thereof in pharmaceutical or nutritional compositions.
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Page/Page column 36
(2015/04/15)
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- CHEMOENZYMATIC SYNTHESIS OF HEPARIN AND HEPARAN SULFATE ANALOGS
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The present invention provides a one-pot multi-enzyme method for preparing UDP-sugars from simple sugar starting materials. The invention also provides a one-pot multi-enzyme method for preparing oligosaccharides from simple sugar starting materials.
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Paragraph 0469; 0470
(2014/09/03)
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- A METHOD FOR PREPARATION OF THE TETRASACCHARIDE LACTO-N-NEOTETRAOSE (LNNT) CONTAINING N-ACETYLLACTOSAMINE
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The present invention relates to a method for preparation of the tetrasaccharide lacto-N-neotetraose (LNnt, formula (I)) especially in large scale, as well as intermediates in the synthesis, a new crystal form (polymorph) of LNnt, and the use thereof in pharmaceutical or nutritional compositions.
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Page/Page column 46
(2011/09/19)
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- Enzymatic supported synthesis of lacto-N-neotetraose using dendrimeric polyethylene glycol
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The lacto-N-neotetraose tetrasaccharide was synthesized on a new dendrimeric support, based on polyethylene glycol. Starting from 1-thio-β-D-lactose, the trisaccharide (2-acetamido-2-deoxy-β-D- glucopyranosyl)-(1→3)-O-β-D-galactopyranosyl-(1→4) -1-thio-β-D-glucopyranose was obtained using Neisseria meningitidis β-(1→3)-N-acetylglucosaminyltransferase according to a soluble synthesis approach, bound on the support and galactosylated using the milk β-(1→4)-galactosyl transferase to give after cleavage the tetrasaccharide lacto-N-neotetraose.
- Renaudie, Laetitia,Daniellou, Richard,Auge, Claudine,Le Narvor, Christine
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p. 693 - 698
(2007/10/03)
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- CRYSTALS OF OLIGOSACCHARIDES AND PROCESSES FOR PREPARATION THEREOF
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The present invention provides crystals of an oligosaccharide useful; for example, as raw materials for or as intermediates of health foods, pharmaceutical compositions, cosmetics, etc. and a process for producing crystals of an oligosaccharide which is suitable for large-scale synthesis or industrialization. That is, the present invention provides a process for producing crystals of an oligosaccharide comprising three or more monosaccharide residues which comprises adding an aqueous solution containing the oligosaccharide comprising three or more monosaccharide residues to a water-miscible organic solvent; a process for producing crystals of an oligosaccharide which comprises adding an aqueous solution containing an oligosaccharide represented by general formula (I): [wherein Gal represents galactose; Glc represents glucose; R1 represents a monosaccharide residue, an amino sugar residue, or a derivative of the monosaccharide residue or the amino sugar residue; R2, R3 and R4, which may be the same or different, each represent a monosaccharide residue, an amino sugar residue, a derivative of the monosaccharide residue or the amino sugar residue, -X(-Y)- (wherein X and Y, which may be the same or different, each represent a monosaccharide residue, an amino sugar residue, or a derivative of the monosaccharide residue or the amino sugar residue) or a single bond; and R5 represents a hydrogen atom, a monosaccharide residue, an amino sugar residue, or a derivative of the monosaccharide residue or the amino sugar residue] to a water-miscible organic solvent; and crystals of an oligosaccharide represented by the above general formula (I).
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- Enzymatic glycosylation of reducing oligosaccharides linked to a solid phase or a lipid via a cleavable squarate linker
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Reducing oligosaccharides were converted into their corresponding glycosylamines, and these were reacted with 3,4-diethoxy-3-cyclobuten-1,2-dione (squaric acid diethyl ester). The resulting derivatives could be linked to amino-functionalized lipids, solids, or proteins. Treatment of the obtained lipid or solid conjugates with aqueous bromine or, alternatively, with ammonia-ammonium borate cleaved the linkage and regenerated the oligosaccharide glycosylamines, which were in turn rapidly hydrolyzed to the reducing oligosaccharides. To demonstrate the usefulness of this linkage in enzymatic oligosaccharide synthesis, lactose was linked to a lipid or a solid phase, the obtained conjugates were then subjected to two enzymatic glycosylations (either consecutively or 'one-pot'). The resulting materials were then cleaved to give, in both cases, the expected reducing tetrasaccharide (lacto-N-neotetraose) in good yield. Copyright (C) 1999 Elsevier Science Ltd.
- Blixt,Norberg
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- Synthesis of lacto-N-neotetraose and lacto-N-tetraose using the dimethylmaleoyl group as amino protective group
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The disaccharide donor O-[2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-(1→4)-3,6-di-O-benzyl-2-deoxy-2-dimethylmaleimido-α,β-D-glucopyranosyl] trichloroacetimidate (7) was prepared by reacting O-(2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl) trichloroaceti
- Aly, Mohamed R.E.,Ibrahim, El-Sayed I.,El Ashry, El-Sayed H.,Schmidt, Richard R.
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p. 121 - 132
(2007/10/03)
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