Streamlined Synthesis and Evaluation of Teichoic Acid Fragments.

Teichoic acids (TAs) are key components of the Gram-positive bacterial cell wall that are composed of alditol phosphate repeating units, decorated with alanine or carbohydrate appendages. Because of their microhetereogeneity, pure well-defined TAs for biological or immunological evaluation cannot be obtained from natural sources. We present here a streamlined automated solid-phase synthesis approach for the rapid generation of well-defined glycosylated, glycerol-based TA oligomers. Building on the use of a "universal" linker system and fluorous tag purification strategy, a library of glycerolphosphate pentadecamers, decorated with various carbohydrate appendages, is generated. These are used to create a structurally diverse TA-microarray, which is used to reveal, for the first time, the binding preferences of anti-LTA (lipoteichoic acids) antibodies at the molecular level.

Teichoic acids: synthesis and applications.

This review describes the developments in the synthesis of teichoic acids (TA) - glycosylated poly(alditolphosphates) - and the application of these fragments in immunological studies. These structurally diverse biopolymers are omnipresent constituents of the Gram-positive bacterial cell wall where they fulfill a variety of vital functions. They have been and continue to be attractive synthetic targets because of their challenging structures and the fact that their microheterogeneity precludes their isolation in single and pure enough form from natural sources. Progress in glycosylation chemistry and the development of effective phosphorylation chemistry has driven TA synthesis over the years, and highly complex and large TA structures can now reliably be targeted. Starting from the first TA synthesis in 1981, this review highlights the progress made in the field over the years. The synthesized TA fragments have been used to unravel their role in immunology and it is described how focused libraries of TAs have been used to discover the active principles of the TA polymers that interact with the innate immune system. Recently, synthetic TA fragments have also found applications as well-defined synthetic antigens for the generation of novel vaccine modalities to combat Gram-positive bacterial infections. It is foreseen that synthetic TA fragments will be valuable tools in the future to unravel the mode of action of these biomolecules at the molecular level. They will be instrumental in discovering and characterizing their designated biological binding partners, be it pattern recognition receptors or carbohydrate binding lectins or biomachinery enzymes. This review thus serves to showcase the potential of organic synthesis for (chemical) biology and immunology.

Total Synthesis of Five Lipoteichoic acids of Clostridium difficile.

The emergence of hypervirulent resistant strains have made Clostridium difficile a notorious nosocomial pathogen and has resulted in a renewed interest in preventive strategies, such as vaccines based on (synthetic) cell wall antigens. Recently, the structure of the lipoteichoic acid (LTA) of this species has been elucidated. Additionally, this LTA was found to induce the formation of protective antibodies against C. difficile in rabbits and mice. The LTA from C. difficile is isolated as a microheterogenous mixture, differing in size and composition, impeding any structure-activity relationship studies. To ensure reliable biological results, pure and well-defined synthetic samples are required. In this work the total synthesis of LTAs from C. difficile with defined chain length is described and the initial biological results are presented.

Synthesis and thermotropic phase behavior of four glycoglycerolipids.

Four glycoglycerolipids with different head groups have been synthesized and their physicochemical properties studied. The lengths of the head groups from a mono-saccharide to a trisaccharide, in addition to the anomeric stereochemistry for the smaller glycoglycerolipids, have been modified. The synthesis has been optimized to avoid glycerol epimerization and to allow up-scaling. The physicochemical properties of the glycoglycerolipids were studied and a strong de-mixing of the gel-phase, depending on the head-group, was observed.

Protection against Staphylococcus aureus by antibody to the polyglycerolphosphate backbone of heterologous lipoteichoic acid.

Type 1 lipoteichoic acid (LTA) is present in many clinically important gram-positive bacteria, including enterococci, streptococci, and staphylococci, and antibodies against LTA have been shown to opsonize nonencapsulated Enterococcus faecalis strains. In the present study, we show that antibodies against E. faecalis LTA also bind to type 1 LTA from other gram-positive species and opsonized Staphylocccus epidermidis and Staphylcoccus aureus strains as well as group B streptococci. Inhibition studies using teichoic acid oligomers indicated that cross-reactive opsonic antibodies bind to the teichoic acid backbone. Passive immunization with rabbit antibodies against E. faecalis LTA promoted the clearance of bacteremia by E. faecalis and S. epidermidis in mice. Furthermore, passive protection also reduced mortality in a murine S. aureus peritonitis model. The effectiveness of rabbit antibody against LTA suggests that this conserved bacterial structure could function as a single vaccine antigen that targets multiple gram-positive pathogens.

Glucose-dependent insulinotropic polypeptide regulates body weight and food intake via GABAergic neurons in mice.

The development of single-molecule co-agonists for the glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) is considered a breakthrough in the treatment of obesity and type 2 diabetes. But although GIPR-GLP-1R co-agonism decreases body weight with superior efficacy relative to GLP-1R agonism alone in preclinical1-3 and clinical studies4,5, the role of GIP in regulating energy metabolism remains enigmatic. Increasing evidence suggests that long-acting GIPR agonists act in the brain to decrease body weight through the inhibition of food intake3,6-8; however, the mechanisms and neuronal populations through which GIP affects metabolism remain to be identified. Here, we report that long-acting GIPR agonists and GIPR-GLP-1R co-agonists decrease body weight and food intake via inhibitory GABAergic neurons. We show that acyl-GIP decreases body weight and food intake in male diet-induced obese wild-type mice, but not in mice with deletion of Gipr in Vgat(also known as Slc32a1)-expressing GABAergic neurons (Vgat-Gipr knockout). Whereas the GIPR-GLP-1R co-agonist MAR709 leads, in male diet-induced obese wild-type mice, to greater weight loss and further inhibition of food intake relative to a pharmacokinetically matched acyl-GLP-1 control, this superiority over GLP-1 vanishes in Vgat-Gipr knockout mice. Our data demonstrate that long-acting GIPR agonists crucially depend on GIPR signaling in inhibitory GABAergic neurons to decrease body weight and food intake.

Synthesis of an alpha-kojibiosyl substituted glycerol teichoic acid hexamer.

In this paper the synthesis of an Enterococcus Faecalis teichoic acid (TA) hexamer is presented. The key kojibiosyl-glycerol phosphoramidite building block was obtained by condensation of thioglucose donors, provided with various protecting groups at the C2 hydroxyl function with an orthogonally protected glycerol acceptor. After selective deprotection, the resulting 1,2-cis-linked pseudodisaccharide acceptor was coupled to an alpha-directing thioglucose donor, giving the corresponding pseudotrisaccharide, which is then transformed to a phosphoramidite synthon. The kojibiosyl-glycerol phosphoramidite in combination with a glycerolphosphoramidite, an aminohexylphosphoramidite and dibenzylglycerol were coupled to a fully protected glycerol TA hexamer, using chemistry that can be amended for future automated synthesis. Global deprotection afforded the target hexamer kojibiosyl-glycerol containing TA (1).

Dually Reactive Long Recombinant Linkers for Bioconjugations as an Alternative to PEG.

Covalent cross-linking of biomolecules can be useful in pursuit of tissue targeting or dual targeting of two receptors on cell surfaces for avidity effects. Long linkers (>10 kDa) can be advantageous for such purposes, and poly(ethylene glycol) (PEG) linkers are most commonly used due to the high aqueous solubility of PEG and its relative inertness toward biological targets. However, PEG is non-biodegradable, and available PEG linkers longer than 5 kDa are heterogeneous (polydisperse), which means that conjugates based on such materials will be mixtures. We describe here recombinant linkers of distinct lengths, which can be expressed in yeast, which are polar, and which carry orthogonal reactivity at each end of the linker, thus allowing chemoselective cross-linking of proteins. A conjugate between insulin and either of the two trypsin inhibitor peptides/proteins exemplifies the technology, using a GQAP-based linker of molecular weight of 17 848, having one amine at the N-terminal, and one Cys, at the C-terminal. Notably, yeast-based expression systems typically give products with mixed disulfides when expressing proteins that are equipped with one unpaired Cys, namely, mixed disulfides with glutathione, free Cys amino acid, and/or a protein homodimer. To obtain a homogeneous linker, we worked out conditions for transforming the linker with mixed disulfides into a linker with a homogeneous disulfide, using excess 4-mercaptophenylacetic acid. Subsequently, the N-terminal amine of the linker was transformed into an azide, and the C-terminal Cys disulfide was reduced to a free thiol and reacted with halo-acetyl insulin. The N-terminal azide was finally conjugated to either of the two types of alkyne-containing trypsin inhibitor peptides/proteins. This reaction sequence allowed the cross-linked proteins to carry internal disulfides, as no reduction step was needed after protein conjugations. The insulin-trypsin inhibitor conjugates were shown to be stabilized toward enzymatic digestions and to have partially retained binding to the insulin receptor.

Development and Characterization of Mouse Monoclonal Antibodies Specific for Clostridiodes (Clostridium) difficile Lipoteichoic Acid.

Clostridiodes (Clostridium) difficile is an anaerobic Gram-positive, spore-forming nosocomial, gastrointestinal pathogen causing C. difficile-associated disease with symptoms ranging from mild cases of antibiotic-associated diarrhea to fatal pseudomembranous colitis. We developed murine monoclonal antibodies (mAbs) specific for a conserved cell surface antigen, lipoteichoic acid (LTA)of C. difficile. The mAbs were characterized in terms of their thermal stability, solubility, and their binding to LTA by surface plasmon resonance and competitive ELISA. Synthetic LTA molecules were prepared in order to better define the minimum epitope required to mimic the natural antigen, and three repeat units of the polymer were required for optimal recognition. One of the murine mAbs was chimerized with human constant region domains and was found to recognize the target antigen identically to the mouse version. These mAbs may be useful as therapeutics (standalone, in conjunction with known antitoxin approaches, or as delivery vehicles for antibody drug conjugates targeting the bacterium), as diagnostic agents, and in infection control applications.

Fluorous linker facilitated synthesis of teichoic acid fragments.

The use of perfluorooctylpropylsulfonylethanol as a new phosphate protecting group and fluorous linker is evaluated in the stepwise solution phase synthesis of a number of biologically relevant (carbohydrate substituted) glycerol teichoic acid fragments. Teichoic acid fragments, up to the dodecamer level, were assembled by means of phosphoramidite chemistry, using a relatively small excess of the building blocks and a repetitive efficient purification procedure of the protected intermediates by fluorous solid phase extraction (F-SPE).

Automated solid phase synthesis of teichoic acids.

This communication describes the first automated solid phase synthesis of teichoic acids (TAs) and the preparation by this method of a number of well-defined TA structures, which were probed for their antigenicity. An opsonophagocytic killing assay revealed a clear TA-length-activity relationship and indicated a promising candidate for future vaccine development.

Stereoselective synthesis of L-guluronic acid alginates.

The glycosylation properties of gulopyranosides have been mapped out, and it is shown that gulose has an intrinsic preference for the formation of 1,2-cis-glycosidic bonds. It is postulated that this glycosylation behaviour originates from nucleophilic attack at the oxacarbenium ion, which adopts the most favourable 3H4 conformation. Building on the stereoselectivity of gulose, a guluronic acid alginate trisaccharide was assembled for the first time by using gulopyranosyl building blocks.