“One Pot Beta-Mannosylations of Glycosyl Hemiacetals”

Imlirenla Pongener ^(a,b), Dionissia A. Pepe ^(a), Eoghan M. McGarrigle ^(a)

^a Centre for Synthesis and Chemical Biology, School of Chemistry

University College Dublin, Belfield, Dublin 4, Ireland

^b Current address: School of Chemical and Physical Sciences, Keele University Staffordshire, UK

E-mail: i.pongener@keele.ac.uk, dionysia.pepe@ucdconnect.ie, eoghan.mcgarrigle@ucd.ie

Despite the tremendous work in the area of chemical glycosylation, the synthesis of 1,2-cis-glycosides, in particular beta-mannosides and beta-rhamnosides still remains challenging. Herein, a highly selective synthesis of beta-mannosides from glycosyl hemi-acetals is reported, following a one-pot chlorination, iodination, glycosylation sequence employing cheap oxalyl chloride, phosphine oxide and an iodide source^(1,2). The present protocol works excellently with a wide range of glycosyl acceptors and with glycosyl donors bearing a range of ethereal protecting groups. The method doesn’t require conformationally restricted donors or directing groups to achieve the high beta-selectivities.

References:

[1] I. Pongener, K. Nikitin and E. M. McGarrigle, Org. Biomol. Chem., 2019, 17, 7531-7535.

[2] I. Pongener, D.A. Pepe and E. M. McGarrigle, manuscript in preparation.

“O-Pair Search with MetaMorpheus for O-glycopeptide Characterization”

Nicholas M. Riley¹, Lei Lu³, Michael R. Shortreed³, Lloyd M. Smith³, Carolyn R. Bertozzi<sup>1,2

1</sup>Department of Chemistry, University of Stanford, Stanford CA 94305

²Howard Hughes Medical Institute, Stanford CA 94305

³Department of Chemistry, University of Wisconsin, Madison, WI 53706

E-mail: nmriley@stanford.edu

Mass spectrometry is the gold standard for interrogating the glycoproteome, enabling the localization of glycans to specific glycosites. Yet, standard approaches for interpreting tandem MS spectra are ill-suited for features inherent to O-glycosylation, including O-glycan heterogeneity and dense stretches of O-glycosylation in serine/threonine rich sequences. Current analysis pipelines are unable to search for multiply glycosylated peptides within reasonable time frames even for simple mixtures of O-glycoproteins, much less for proteome-scale experiments. Moreover, current software tools for O-glycopeptide identification fail to capitalize on modern MS-acquisition methods, e.g., combinations of collision-based and electron-based dissociation within the same analysis, which offer complementary coverage of both peptide backbone and glycan fragmentation. Existing tools also lack the ability to confidently localize glycosites within multiply glycosylated O-glycopeptides.

Here we describe the O-Pair Search strategy implemented in the MetaMorpheus platform. Using paired collision- and electron-based dissociation spectra collected for the same precursor ion, O-Pair Search identifies O-glycopeptides in four steps: 1) rapid identification of peptide candidates using a fragment ion indexing search strategy, 2) determination of possible O-glycans present on peptide candidates using combined glycan total masses, 3) localization of individual O-glycans to specific O-glycosites using graph theory for spectra from electron-driven dissociation, and 4)  calculation of probability-based localization scores for each localized glycosite, a first for glycopeptides.

With O-Pair Search, we show that search times for O-glycopeptides from simple mixtures can be reduced by >2000x over the most widely used commercial glycopeptide search tool (Byonic), requiring <1 min with MetaMorpheus compared to the >12 hours using Byonic. Additionally, O-Pair Search identifies more O-glycopeptide identifications than Byonic and reports localization levels that indicate if all (Level 1), at least one (Level 2), or none (Level 3) of the O-glycosites are confidently localized – a feature previously unavailable on any other platform. We further demonstrate the utility of O-Pair Search by performing searches using larger glycan databases, larger protein databases, and O-glycoproteomic data from complex mixtures (i.e., searches that are not practical in Byonic).

“Exploratory N-protecting group manipulation for the total synthesis of zwitterionic Shigella sonnei oligosaccharides”

Debashis Dhara^[a]  and Laurence A. Mulard^[a] *

^[a] Institut Pasteur, Unit de Chimie des Biomolcules, CNRS UMR 3523, 28 rue du Dr Roux, 75015 Paris, France

E-mail: dhara.debashis@pasteur.fr

Shigellosis or bacillary dysentery is caused by the enteroinvasive bacteria Shigella. An estimated half a million shigellosis cases occur only in the United States each year.^[1]  Shigella flexneri together with Shigella sonnei are responsible for 90% of shigellosis worldwide. A Phase III clinical trials has demonstrated that detoxified Shigella sonnei lipopolysaccharide-conjugate vaccine is safe and immunogenic against S. sonnei infection in young adults and children older than 3 years of age.^[2] As a possible alternative to the use of the detoxified lipopolysaccharide, we have engaged in developing a glycoconjugate vaccine encompassing fully synthetic oligosaccharides covalently attached to a carrier protein.

S. sonnei surface polysaccharides are made of a unique zwitterionic polysaccharides (ZPSs) featuring a disaccharide repeating unit made of two 1,2-trans linked rare aminodeoxy sugars, a 2-acetamido-2-deoxy-L-altruronic acid (L-AltpNAcA) and a 2-acetamido-4-amino-2,4,6-trideoxy-D-galactopyranose (AAT).^[3]  The synthesis of fragments of these zwitterionic polysaccharides was achieved through a fine-tuned N-protecting group selection and manipulation at the conformational flexible of L-AltpNAcA within disaccharide building blocks.  A convergent synthesis of zwitterionic tetra-, hexa- and octa-saccharides made of such two rare aminodeoxysugars will be presented.^[4]

References
[1] US Centers for Disease Control and Prevention. "Shigella – Shigellosis". https://www.cdc.gov/shigella/general-information.html#resistance
[2] Passwell, J. H.; Ashkenazi, S.; Miron, D.; Ramon, R.; Farzam, N.; Lerner-Geva, I.; Levi, Y.; MHA and the israel Shigella study group.; Chu, C., Shiloach, J.; Robbins, J. B.; Schneerson R.; Safety and immunogenicity of Shigella sonnei-CRM9 and Shigella flexneri type 2a-rEPAsucc conjugate vaccines in one- to four-year-old children. Pediatr. Infect. Dis. J. 2003, 22, 701-706.
[3] Pfister, H. B.; and Mulard, L. A.; Synthesis of the zwitterionic repeating unit of the O-antigen from Shigella sonnei and chain elongation at both ends. Org. Lett. 2014, 16, 4892-4895.
[4] Dhara, D.; and Mulard, L. A.; Exploratory N-protecting group manipulation for the total synthesis of zwitterionic Shigella sonnei oligosaccharides. Chem. Eur. J. 2020, (accepted for publication).

“Overcoming the challenges in bifidobacteria exopolysaccharides isolation”

Michela Ferrari¹, Marjolein Oerlemans², Paul de Vos², Marthe Walvoort^1
1Stratingh Institute for Chemistry, University of Groningen
²Department of Pathology and Medical Biology, University Medical Center Groningen

Bacterial exopolysaccharides (EPS) are high-molecular weight polysaccharides that are loosely attached to the cell surface or excreted in the environment. Their molecular structures vary among different strains, and can contain unusual carbohydrates and glycosidic linkages that are not found in eukaryotes. EPS have been studied extensively in the past years as they are part of bacterial biofilms, and combine interesting technological properties with promising effects on human health. Bifodobacteria are anaerobic human commensals mainly located in the gut and other mucosal niches. These gram-positive bacteria are EPS producers and since their discovery, have been in the spotlight due to their potential beneficial properties on health. In the past years isolation of bifidobacteria EPS has been limited by low yields, long fermentation times and contamination by enriched media. In the current study we present a general protocol that can be used to face the challenges encountered performing the isolation and characterization of bifidobacteria EPS.

“Anti-glycan antibodies in colorectal cancer diagnosis”

Aleksei Tikhonov¹, Nikolay Kushlinskii², Alla Rubina¹

¹Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

²«N.N.Blokhin National Medical Research Center of Oncology» of the Ministry of Health of Russia

E-mail: alex.tihonoff@gmail.com

Colorectal cancer (CRC) at the initial stages is usually asymptomatic, and it leads to the fact that the disease is often detected at late stages. Therefore, the problem of early CRC diagnosis using minimally invasive methods is currently urgent. In this work, we studied antibodies to various cancer-associated glycans in human blood for the identification of potential cancer biomarkers.

At the moment, a huge number of markers associated with tumor progression are known, but most of them do not have a high enough specificity for diagnosing a particular disease. Thus, the idea of a single biological marker, the so-called "golden bullet", is theoretically very attractive, but in reality, it turns out to be practically unrealizable. Therefore, an approach based on the detection of several markers simultaneously seems reasonable.

We developed a multiplex FLISA-based method using hydrogel microarrays for the detection of anti-glycan antibodies and demonstrated the possibility of using such an approach for the CRC diagnosis and for characterization of different CRC cohorts using different levels of specific anti-glycan antibodies.