Synthesis of Oligodeoxyribo‐ and Oligoribonucleotides According to the H‐Phosphonate Method

Oligonucleotides can be synthesized by condensing a protected nucleoside H‐phosphonate monoester with a second nucleoside in the presence of a coupling agent to produce a dinucleoside H‐phosphonate diester. This can then be converted to a dinucleoside phosphate or to a backbone‐modified analog such as a phosphorothioate or phosphoramidite. This unit discusses four alternative methods for synthesizing nucleoside H‐phosphonate monoesters. The methods are efficient and experimentally simple, and use readily available reagents. The unit describes the activation of the monoesters, as well as competing acylation and other potential side reactions.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143594/1/cpnc0304.pd

Deoxyribo‐ and Ribonucleoside H‐Phosphonates

Most methods for preparing H‐phosphonate monoesters suffer from variable yields and are often incompatible with common protecting groups used in oligonucleotide synthesis. This unit describes four procedures that consistently give high yields of the desired products. Taken together, they provide an arsenal of phosphonylation prodecures that it compatible with most common protecting groups.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143703/1/cpnc0206.pd

Syntheses of the Biological Repeating Units of Salmonella Serogroups A, B, and D, 0-Antigenic Polysaccharides

The immunogenic polysaccharide part of the Salmonella lipopolysaccharide of the cell walls of Salmonella bacteria belonging to serogroups A, B, and D, is depicted below. 2-OAC , 6 -D i d e o x y -a -D -H e x p -Dideoxy -a-D-Hexp Syntheses of the following three tetrasaccharides are described : p-trifluoroacetamidophenyl 0-(3,6- 0-a-D-galactopyranoside, in which the 3,6-dideoxyhexopyranosyl group is abequosyl (28), paratosyl galaCtOpyranOSide with all hydroxy-groups protected but the one at C-3 in the mannosyl unit. In the various glycosylations, halide-ion catalysis or silver triflate-promotion was used in the construction of 1,2-cis-glycosidic bonds from glycosyl halides with a non-participating group in the 2-position. Silver triflate was used as promotor in the construction of 1,2-trans-glycosidic bonds from glycosyl halides carrying a participating acyl group in the 2-position. In our programme aimed at the synthesis of oligosaccharides which are part of 0-antigenic Salmonella polysaccharides, we have previously synthesized glycosides of the disaccharides 3-0-(3,6-dideoxy-a-~-ribo-hexopyranosyl)-a-D-mannopyranose,1*2 3-04 3,6-dideoxy-a-~-xylo-hexopyranosyl-)-a-~-mannopyran~se,~ 3-0-(3,6-dideoxy-a-~-arabino-hexopyranosy1)-a-D-mannopyranose," and 3-0-( 3,6-dideoxy-a-~-xylohexopyranosyl-)-a-~-rhamnopyranose.~ Also synthesized were the disaccharides 4-0-(a-~-mannopyranosy~-)-a-~-rhamnopyranose and 4-O-( P-~-mannopyranosyl-)-a-~-rhamnopyranose and the trisaccharide 0-a-D-galactopyranosyl-

Non-Glycosidically Linked Pseudodisaccharides: Thioethers, Sulfoxides, Sulfones, Ethers, Selenoethers, and Their Binding to Lectins

Hydrolytically stable non-glycosidically linked tail-to-tail pseudodisaccharides are linked by a single bridging atom remote from the anomeric centre of the constituent monosaccharides. Some such pseudodisaccharides with sulfur or oxygen bridges were found to act as disaccharide mimetics in their binding to the Banana Lectin and to Concanavalin A. A versatile synthetic route to a small library of such compounds is described.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/69166/1/1951_ftp.pd

The Development of Visible‐Light Photoredox Catalysis in Flow

Visible‐light photoredox catalysis has recently emerged as a viable alternative for radical reactions otherwise carried out with tin and boron reagents. It has been recognized that by merging photoredox catalysis with flow chemistry, slow reaction times, lower yields, and safety concerns may be obviated. While flow reactors have been successfully applied to reactions carried out with UV light, only recent developments have demonstrated the same potential of flow reactors for the improvement of visible‐light‐mediated reactions. This review examines the initial and continuing development of visible‐light‐mediated photoredox flow chemistry by exemplifying the benefits of flow chemistry compared with conventional batch techniques.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106928/1/351_ftp.pd

Characterization of the LM5 pectic galactan epitope with synthetic analogues of β-1,4-d-galactotetraose

Plant cell wall glycans are important polymers that are crucial to plant development and serve as an important source of sustainable biomass. The study of polysaccharides in the plant cell wall relies heavily on monoclonal antibodies for localization and visualization of glycans, using e.g. Immunofluorescent microscopy. Here, we describe the detailed epitope mapping of the mab LM5 that is shown to bind to a minimum of three sugar residues at the non-reducing end of linear beta-1,4-linked galactan. The study uses de novo synthetic analogs of galactans combined with carbohydrate microarray and competitive inhibition ELISA for analysis of antibody-carbohydrate interactions

Synthesis of 4-methylumbelliferyl α-d-mannopyranosyl-(1→6)-β-d-mannopyranoside and development of a coupled fluorescent assay for GH125 exo-α-1,6-mannosidases

Certain bacterial pathogens possess a repertoire of carbohydrate processing enzymes that process host N-linked glycans and many of these enzymes are required for full virulence of harmful human pathogens such as Clostridium perfringens and Streptococcus pneumoniae. One bacterial carbohydrate processing enzyme that has been studied is the pneumococcal virulence factor SpGH125 from S. pneumoniae and its homologue, CpGH125, from C. perfringens. These exo-α-1,6-mannosidases from glycoside hydrolase family 125 show poor activity toward aryl α-mannopyranosides. To circumvent this problem, we describe a convenient synthesis of the fluorogenic disaccharide substrate 4-methylumbelliferone α-d-mannopyranosyl-(1→6)-β-d-mannopyranoside. We show this substrate can be used in a coupled fluorescent assay by using β-mannosidases from either Cellulomonas fimi or Helix pomatia as the coupling enzyme. We find that this disaccharide substrate is processed much more efficiently than aryl α-mannopyranosides by CpGH125, most likely because inclusion of the second mannose residue makes this substrate more like the natural host glycan substrates of this enzyme, which enables it to bind better. Using this sensitive coupled assay, the detailed characterization of these metal-independent exo-α-mannosidases GH125 enzymes should be possible, as should screening chemical libraries for inhibitors of these virulence factors

A colanic acid operon deletion mutation enhances induction of early antibody responses by live attenuated salmonella vaccine strains

Colanic acid (CA) is a common exopolysaccharide produced by many genera in the Enterobacteriaceae. It is critical for biofilm formation on HEp-2 cells and on chicken intestinal tissue by Salmonella. In this study, we generated different CA synthesis gene mutants and evaluated the immune responses induced by these mutants. One of these mutations, Δ(wza-wcaM)8, which deleted the whole operon for CA synthesis, was introduced into two Salmonella vaccine strains attenuated by auxotrophic traits or by the regulated delayed attenuation strategy (RDAS). The mice immunized with the auxotrophic Salmonella vaccine strain with the deletion mutation Δ(wza-wcaM)8 developed higher vaginal IgA titers against the heterologous protective antigen and higher levels of antigen-specific IgA secretion cells in lungs. In Salmonella vaccine strains with RDAS, the strain with the Δ(wza-wcaM)8 mutation resulted in higher levels of protective antigen production during in vitro growth. Mice immunized with this strain developed higher serum IgG and mucosal IgA antibody responses at 2 weeks. This strain also resulted in better gamma interferon (IFN-γ) responses than the strain without this deletion at doses of 10(8) and 10(9) CFU. Thus, the mutation Δ(wza-wcaM)8 will be included in various recombinant attenuated Salmonella vaccine (RASV) strains with RDAS derived from Salmonella enterica serovar Paratyphi A and Salmonella enterica serovar Typhi to induce protective immunity against bacterial pathogens

An atypical interaction explains the high-affinity of a non-hydrolyzable S-linked 1,6-α-mannanase inhibitor

The non-hydrolyzable S-linked azasugars, 1,6-α-mannosylthio- and 1,6-α-mannobiosylthioisofagomine, were synthesized and shown to bind with high affinity to a family 76 endo-1,6-α-mannanase from Bacillus circulans. X-ray crystallography showed an atypical interaction of the isofagomine nitrogen with the catalytic acid/base. Molecular dynamics simulations reveal that the atypical binding results from sulfur perturbing the most stable form away from the nucleophile interaction preferred for the O-linked congener

Benzylation of hydroxy groups with tertiary amine as a base

The benzylation of hydroxy groups in the presence of tertiary amines is described. A mixture of an alcohol and a benzyl halide afforded the corresponding benzyl ether in good to excellent yields in the presence of diisopropylethylamine. The importance of solventless conditions was observed. The reaction showed high tolerance to many functional groups including benzoate, even at a reaction temperature of 150 °C. Sodium iodide was found to be an efficient catalyst to accelerate the reaction