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Division/Committee: [CARB] Division of Carbohydrate Chemistry

This Symposium will encompass recent developments in all areas of
Tuesday

Tuesday
Diverse pathogens expose on their surface a dense, often highly conserved array of glycan structures that exert a protective function against the host’s immune defence and are essential for their pathogenicity. Typical examples are lipopolysaccharides (LPS) of Gram-negative bacteria, the polysaccharide coat (capsular polysaccharides, CPS) of encapsulated bacteria, and glycoproteins/glycolipids specifically expressed on the surface of viruses and parasites.
All these glycoforms are capable of interacting with the immune system as epitopes inducing the production of carbohydrate-specific antibodies. They, therefore, represent attractive targets for vaccine design. In this regard, glycoconjugate vaccines, based on the covalent linking to immunogenic proteins of glycan structures uniquely exposed on the surface of pathogens, give rise to conventional B cell-mediated immunological memory, and exert a satisfying immunological activity even among persons in high-risk groups. Several conjugate versions of polysaccharide vaccines are now either commercially available or in development. Additional benefits are expected from the development of a new generation of glycoconjugate vaccines based on the use of synthetic saccharide antigens. The chemical synthesis of oligosaccharides provides homogeneous and well defined molecules with built-in chemical terminal functionalities suitable for conjugation to carrier proteins. Furthermore, only chemical synthesis allows modification of glycoepitopes to break immune tolerance and improve their immunogenicity, as well as to increase their chemical stability. Accordingly, in the past decade, a variety of oligosaccharides resembling or mimicking bacterial carbohydrates have been synthesized by us and other groups, protein conjugated and proved to elicit protective antibodies in animal models.
Numerous bacterial polysaccharides are phosphoglycans, in which the phosphodiester linkages connecting the saccharide repeating units are often immunologically active components and crucial virulence determinants.
In the present communication, some significant examples of the contribution of our laboratory to the synthesis and immunoevaluation of bacterial phosphoglycans will be illustrated and discussed, showing how synthetic organic chemists can play a leading role in the design and development of new and more efficacious vaccines against infectious diseases.
Tuesday
Innate Immunity is the first defense line in multicellular organisms against internal of external threats. It acts through inflammation, triggered by the recognition of specific Pathogen or Damage Associated Molecular Patterns (PAMPs or DAMPs) by specific pattern-recognition receptors (PRRs). Toll-Like Receptor 4 (TLR4) is one of the most important PRRs, and it responds to gram-negative bacteria lipopolysaccharide (LPS).

TLR4 modulation is emerging as an important therapeutic approach in several clinical settings: TLR4 inhibition has a potent anti-inflammatory effect; on the other hand, TLR4 mild activation can be used to stimulate immunity in vaccine adjuvants or to develop cancer immunotherapeutic drugs.

We present here rationally designed lipid A analogues based on a monosaccharide structure that are active in binding MD-2/TLR4, thus activating or inhibiting LPS/TLR4 or DAMP/TLR4 signalling. We also present synthesis optimization of TLR4 modulators, with the aim of producing versatile synthetic intermediates and reducing the number of synthetic steps to efficiently scale the synthesis up for industrial purposes.

Tuesday
Siglecs (Sialic acid-binding immunoglobulin type lectins) are a family of cell surface transmembrane receptors belonging to I-type lectins, predominantly expressed by immune cells. Individual family members exhibit preferences for sialosides of various linkages to underlying glycan motifs, but the physiological ligands they interact with are largely unknown.
Many Siglecs, such as Siglec-2, Siglec-7 and Siglec-10, are inhibitory receptors involved in the down-regulation of cell signalling upon the interaction with sialylated glycans that act as determinants of self. Interestingly, clinically relevant pathogens have the ability to decorate their surface with glycans that mimic self-associated molecular patterns, bind to inhibitory Siglecs, and escape immune surveillances.
Thus, Siglecs are nowadays considered glyco-immune checkpoints and exhibit a great therapeutic potential for the treatment of autoimmune, neurodegenerative and cancer diseases.
In this context, we investigated the molecular mechanisms underlying sialoglycans recognition by Siglecs using a combination of biophysical, spectroscopic and computational approaches, with the aim to carry out a dynamic characterization of their interactions in solution. NMR spectroscopy, and in particular ligand-based NMR techniques including STD- NMR and tr-NOESY, were used to evaluate the interacting epitope and the bioactive conformation of sialoglycans in solution. Homology modeling, docking and MD studies, together with CORCEMA-ST protocol, were implemented to obtain and validate 3-D ligand/receptor complexes, highlighting the crucial interactions between the binding partners. Comprehensively, our outcomes have improved the knowledge of the molecular interaction occurring between Siglecs and sialyloglycans, providing a structural point of view for the design and development of high-affinity ligands able to control the receptor functionality.
Tuesday
The stereocontrolled chemical synthesis of sialic acid containing glycoconjugates has been historically one of the most challenging tasks in the field. Today, thanks to the effort of several research groups and especially to the discovery of the effect of substituents at C-5, complex synthetic targets are more accessible. Nevertheless, current methodologies are still limited by multistep protecting groups manipulations and/or low temperatures required in sialylations.
Contrarily to the mainstream in carbohydrate synthesis, little is known about O-protecting group manipulations and their effects in sialylations. As a part of an ongoing investigation toward the effect of protecting groups at C-4 and the glycerol chain, herein we report the application of novel O-protecting groups in sialylation reactions.
Tuesday

Tuesday

Tuesday
Glycoconjugate vaccines are an important and successful countermeasure to control and prevention of infectious disease. Elucidating the shortest portion of a polysaccharide able to bind to specific functional monoconal antibodies is key for optimal vaccine design, particularly when short synthetic carbohydrates are used. Recently we performed structural studies to identify the minimal epitope of group B Streptococcus type III polysaccharide (GBS PSIII), a leading cause of invasive infections in pregnant women, newborns, and elderly people, and the capsule is a major virulence factor targeted for vaccine development. GBS PSIII epitope has been historically considered the prototype of a complex conformational carbohydrate epitope. Applying an integrated approach based on competitive ELISA/Surface Plasmon Resonance/Saturation Transfer NMR/X-ray we elucidated a structural epitope consisting of five residues from a single repeating unit, and the GlcNAc moiety of the next consecutive repeat unit, where sialic acid is clearly engaged in antibody binding. Starting from this finding we designed a conjugate vaccine from a synthetic hexasaccharide which was capable to elicit in mice functional antibodies comparably to a polysaccharide conjugate.
Likewise, we mapped the structural epitope of Neisseria meningitidis serogroup A (MenA), a Gram-negative encapsulated bacterium responsible for epidemic meningitis in the sub-Saharan region of Africa, termed meningitis belt. Despite multivalent and monovalent conjugate vaccines against MenA are now available, the structural minimal epitope of MenA polysaccharide is still unknown. MenA capsular polysaccharide (CPS) consists of (1→6) linked mannosamine phosphate repeating units with O-acetylation predominantly at position 3. We found that the epitope is a linear trimer with alternated 3-4-3 acetylation pattern. Based on this information we designed an acetylated glycomimetic MenA vaccine where the natural polysaccharide is replaced by a carba-analogue randomly acetylated at position 3 and 4. This conjugate, had a higher stability to hydrolysis compared to the natural polymer, which is expected to ensure better storage. Immunization of mice with the acetylated carbaMenA conjugate induced high levels of functional antibodies. Efforts in understanding the structural basis of recognition of oligosaccharide epitopes are the foundations for future development of synthetic carbohydrate based vaccines.
Tuesday

In spite of many methods developed for the synthesis of glycans, glycosyl halide donors discovered by Michael continue to find wide application. Under classical Koenigs-Knorr reaction conditions, a glycosyl bromide (or chloride) donor is coupled with a glycosyl acceptor (alcohol, ROH) in the presence of silver oxide (or carbonate). This reaction is slow, and even glycosidations of reactive, per-benzylated donors require many hours (or even days) to produce the respective glycoside products. This reaction is particularly sluggish with less reactive per-benzoylated bromides.

A recent discovery of a cooperative catalysis comprising a silver salt and an acid led a dramatic improvement in the way glycosyl halides are glycosidated. Excellent yields have been achieved, but the stereoselectivity achieved with 2-O-benzylated donors was poor. Reported herein is our effort to refine the stereoselectivity of the cooperatively-catalyzed galactosylation reaction. Careful optimization of the reaction conditions along with studying effects of the remote protecting groups led to excellent stereocontrol of α-galactosylation of a variety of glycosyl acceptors with differentially protected glycosyl donors. The developed method has the following advantages in comparison with the known methods: no specialized protecting groups are required therefore synthesis of donors is straightforward. In addition, the reaction is swift and can be conducted at ambient temperature without losing stereoselectivity and the promoter systems are stable and commonly available.
Tuesday
Lipopolysaccharides (LPS) from gut commensal bacteria trigger immunomodulatory responses on the basis of their structures. However, only a few gut commensal LPS have been structurally elucidated so far. Therefore, the molecular motifs crucial for LPS−host interactions at the gut level remain obscure. In this communication, I will focus on the LPS of two commensals of the human intestine: Bacteroides vulgatus and Alcaligenes faecalis. I will show that B. vulgatus LPS does not induce proinflammatory cytokines release and that its administration is enough to reestablish intestinal immune homeostasis in a mouse model for experimental colitis. I will also present that the LPS structural characterization revealed an unprecedented structure based on a hypo-acylated and mono-phosphorylated lipid A, a galactofuranose-containing core OS, and an O-antigen built up of mannose and rhamnose. To this particular structure corresponds an intriguing ability, in human in vitro models, to produce antiinflammatory cytokines and to induce the synergistic activation of TLR4- and TLR2-mediated signaling pathways. As for A. faecalis, this is the sole Gram-negative inhabiting gut lymphoid tissues, Peyer’s patches (PPs), which are the largest sites for the initiation and regulation of intestinal IgA responses. We already showed that Alcaligenes LPS maintain a homeostatic environment in PPs, without triggering any harmful response. Acting as such, this LPS would take part in the host immune vigilance through the production of IgA, which in turn might favor their persistence in PPs. Here I will highlight that also A. faecalis LPS has an unreported structure with a mono-phosphorylated core OS, which contains a huge number of N-acetyl hexosamines. The O-antigen is a xylosylated rhamnan chain while the lipid A is a mixture of tetra- to hexa-acylated species. Finally, I will show that these differently acylated lipid A have been synthesized and their immunological properties tested, revealing that only the hexa-acylated one is able to induce NF-κB activation in TLR4-expressing cells, which was however weaker than upon stimulation by E. coli LPS.

Tuesday
The surface of all cells is surrounded by a diverse mixture of complex carbohydrate structures called glycans. The interactions of glycans with proteins is central to a diverse range of physiological and pathological processes including cell-cell communication, molecular recognition, immunological responses, infectious diseases and cancers. However, identifying the precise endogenous glycan structures involved in these interactions and the mechanisms by which they elicit function is a major challenge. The heterogeneity and complexity of cell-surface glycans, coupled with their biosynthesis not being template-driven, make it difficult to elucidate biological function by traditional approaches. As a result, novel chemical biology tools are needed to meet the demand for new information and advance our understanding of the function of these important biomolecules.
In this presentation, we will describe an interdisciplinary approach to tackle this challenge of studying glycan function in cells. We use chemo-enzymatic synthetic strategies to prepare carbohydrate-based probes and biologically relevant complex glycan structures. These probes or glycans are then installed on cells using an enzymatic-based cell-surface engineering methodology to investigate how these structures interact with glycan-binding proteins and modulate cell function, allowing for structure-function analysis of glycans within the context cells. Chemo-enzymatic approaches to synthesize chemical probes and glycan structure will be discussed. In addition, studies using cell-surface engineering to examine the role of glycans and glycoproteins in immunological responses and virus interactions will also be described. These techniques will facilitate the identification of functional glycan ligands to glycan-binding proteins, as well as glycan biomarkers and targets for therapeutics to facilitate the development of glycan-based strategies for combatting disease.
Tuesday

Division/Committee: [MEDI] Division of Medicinal Chemistry

New treatments to address COVID-19 pandemia
Tuesday

Tuesday
Currently, we are immersed in a pandemic caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which severely threatens public health worldwide. This is a highly-pathogenic human coronavirus (CoV) first reported in Wuhan, China, where a pneumonia of unknown cause was detected in December 2019.

The shortage of really effective drugs or vaccines to treat the serious disease caused by this coronavirus, COVID-19, has prompted scientists to intensify their work. Scientific accumulated knowledge for years has shared in an unprecedented inter- and multi-disciplinary fashion. Drug discovery community has focused the efforts on identify pharmacological targets and therapeutic candidates that finally can provide the desired treatment to combat the pandemia.

This communication will present some of the main virus and host-based targets that can guide the efforts of medicinal chemistry to discover new drugs together with a description of the new ones recently discovered. Furthermore, results from our group focused on target and ligand-based drug discovery using chemical libraries will be also described.

The joint work of the scientific community, both the private and the public sectors, will undoubtedly help to find a solution to the current serious global health problem.
Tuesday
Remdesivir (GS-5734; RDV) is a phosphoramidate prodrug of modified adenosine nucleotide analog with a broad-spectrum antiviral activity against multiple RNA viruses including filoviruses, paramyxoviruses, flaviviruses and coronaviruses. RDV is intracellularly metabolized to the active nucleoside triphosphate form that directly inhibits viral replication following its incorporation into viral RNA by viral RNA-dependent RNA polymerases. The molecular mechanism leading to the termination of viral RNA synthesis as well as key structural interactions with the target viral RNA polymerases have been established. Multiple independent studies have established the antiviral activity of RDV against SARS-CoV-2 both in vitro and in animal models. In several randomized controlled clinical trials, 5- to 10-day course of treatment with RDV administrated once-daily by intravenous infusion has demonstrated efficacy in hospitalized COVID-19 patients by significantly reducing disease progression and accelerating time to recovery. In October 2020, RDV has been approved by US FDA as the first treatment for COVID-19 in hospitalized patients. RDV is currently being tested in combinations with a variety of other agents including anti-inflammatory drugs as well as neutralizing antibodies to potentially further enhance the clinical impact in hospitalized settings. In addition, a newly developed inhaled formulation of RDV is undergoing a Phase2 evaluation in out-patient settings with a goal to prevent disease progression and reduce hospitalization following COVID-19 diagnosis.
Tuesday
Small molecule inhibition of the viral main protease (Mpro) has been a successful anti-viral therapeutic strategy in HIV and HCV. Structural insight on the SARS-CoV-2 Mpro and previous small molecule experience with intravenous SARS-CoV-1 inhibitors gave a starting point for an oral Mpro inhibitor program in response to the COVID-19 outbreak. The discovery of an oral SARS-CoV-2 Mpro inhibitor that displays potent anti-viral activity will be described. The lead molecule from the program is targeted to start clinical studies in 1Q21.
Tuesday
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the resulting coronavirus disease 2019 (Covid-19) have afflicted tens of millions of people since it was declared a pandemic by the World Health Organization on March 11, 2020. Development and roll out of safe and effective prophylactic vaccines are urgently needed to contain the pandemic and stem the devastating medical, economic, and social consequences of COVID-19. In an ongoing multinational, placebo-controlled, observer-blinded, pivotal efficacy trial, Pfizer-BioNTech randomly assigned participants initially 18 years of age or older in a 1:1 ratio to receive two doses of either placebo or BNT162b2 (30 μg per dose). BNT162b2 is a lipid nanoparticle-formulated, nucleoside-modified RNA vaccine that encodes a prefusion stabilized, membrane-anchored SARS-CoV-2 full-length spike protein. The primary end points were safety and efficacy of the vaccine against laboratory-confirmed Covid-19. A total of 43,548 participants underwent randomization. There were 8 cases of Covid-19 with onset at least 7 days after the second dose among participants that received BNT162b2 and 162 cases among placebo recipients. BNT162b2 was determined to be 95% effective in preventing Covid-19 (95% credible interval, 90.3 to 97.6). The safety profile of BNT162b2 was characterized by short-term, mostly mild-to-moderate pain at the injection site, fatigue, and headache. The incidence of serious adverse events was low and similar in the vaccine and placebo groups. The results provide proof of concept that RNA-based vaccines are a promising new vaccine approach to protect humans against infectious diseases. This rigorous demonstration of safety and efficacy, less than 11 months after the SARS-CoV-2 genetic sequence was released, provides a practical demonstration that RNA-based vaccines can be powerful to combat pandemics. With the appearance of new SARS-CoV2 variants, data are now being generated to assess the ability of vaccine induced antibodies to neutralize these emerging variants.
Tuesday
A new class of drugs wherein cells are programmed with synthetic messenger RNAs (mRNAs) to make any desired protein (e.g., cytoplasmic, intraorganelle, membrane-bound, secreted) is an emergent technology with tremendous promise. The ability to simultaneously deliver multiple mRNAs species enables production of multiprotein complexes in their native state. mRNA therapeutics already in or soon to enter the clinic include mRNA-based vaccines (both prophylactic and therapeutic), pro-inflammatory cytokines as anticancer agents, an angiogenic factor for blood vessel regrowth in damaged heart muscle, and protein replacement therapies for treatment of metabolic diseases. Nonetheless, how to combat mRNA’s inherent chemical and biological lability, how to direct therapeutic mRNAs to desired cell types, and how to enable repeat dosing without eliciting adverse immune reactions remain challenges for the field. I will discuss recent progress at Moderna in overcoming these challenges, with particular emphasis on our development of new technologies optimized for functional mRNA delivery.
Division/Committee: [CARB] Division of Carbohydrate Chemistry

This virtual symposium aims to highlight the power of chemical biology, biochemistry, and glycobiology, and to bring the community together to address critical challenges in Glycoscience. This topic is especially important given the recent name change of our CARB division. We plan to invite both young and established investigators in related fields to present talks with a goal to expand the visibility of our division and to attract a diverse and wider audience.
Tuesday

Tuesday
Cell wall peptidoglycan is an essential interface between a bacterium and its surroundings. This dynamic structure accommodates growth and division while protecting against environmental insults. Because it is essential for viability and is composed of molecules that are not present in eukaryotic cells, the bacterial cell wall has been a fruitful target for antibiotic development. The peptidoglycan biopolymer consists of a glycan backbone and cross-linked peptides. The clinical success of cross-link-inhibiting beta-lactam antibiotics illustrates the essentiality of these linkages for cell wall integrity, but the presence of multiple, seemingly redundant transpeptidases in many bacterial species makes it challenging to determine cross-link function precisely. We recently developed a method for introducing synthetic crosslinks into the cell wall of live bacteria. The fluorescent “staples” specifically rescue the model organism Escherichia coli from broad-spectrum beta-lactams. Chemically-induced cross-linking complements genetic and small molecule perturbations as an independent means of investigating the role of cell wall connectivity in bacterial physiology.
Tuesday
The antitumor efficacy of cancer immunotherapy has been correlated with specific species within the gut microbiota. However, molecular mechanisms by which these microbes affect host response to immunotherapy remain elusive. Here we show that specific members of the bacterial genus Enterococcus can promote anti-PD-L1 immunotherapy in mouse tumor models. The active enterococci express and secrete orthologs of the NlpC/p60 peptidoglycan hydrolase SagA that generate immune-active muropeptides. Expression of SagA in non-protective E. faecalis was sufficient to promote antitumor activity of clinically approved checkpoint targets, and its activity required the peptidoglycan sensor Nod2. Notably, SagA-engineered probiotics or synthetic muropeptides also promoted checkpoint inhibitor antitumor activity. Our data suggest that microbiota species with unique peptidoglycan remodeling activity may enhance immunotherapy and could be leveraged for next-generation adjuvants.
Tuesday
The human gut microbiota is a complex microbial community, where trillions of microbes including bacterial and fungi co-exist together. Inevitably, the growths and activities of the microbes are intricately regulated by other members in the microbiota milieu. Clinically, an important observation is that the use of broad-spectrum beta-lactam antibiotics in patients significantly increases the incidences of fungal infections by Candida albicans, which is a major fungi of the human microbiota. While the long-standing belief is that the removal of gut bacteria by beta-lactam drugs creates a niche that favors fungi growth and dissemination, this explanation fails to reconcile with several recent findings on Candida albicans invasions, suggesting different underlying modes of bacterial-fungal interactions in the host microbiota. In this work, we have unravelled that administrations of beta-lactam antibiotics lead to a significant peptidoglycan (PGN) storm in the host microbiota milieu that strongly contributes to Candida albicans hyphal growth and dissemination in vivo. This work has important implications on the appropriate choice of antibiotics to reduce associated fungal infections as well as highlighting potential avenues for development of antifungal therapeutics.
Tuesday

Tuesday
On antigen presenting cells, lipid antigens, including glycolipids such as α-GalCer are recognized by CD1d, which is a non-polymorphic MHC class I-like molecule. Complexes of glycolipid ligands and CD1d are recognized by T cell receptors (TCR) on NKT cells and induce the secretion of various cytokines, such as helper-T (Th)1 and Th2 cytokines. We have synthesized some series of glycolipids, including glycerol- and ceramide-type compounds, and investigated the modulation of their cytokine-biasing responses. Although Th2-biasing CD1d ligands are attractive potential candidates for adjuvants and therapeutic drugs for autoimmune diseases, only a limited number of potent ligands have been reported. We have recently identified a series of novel Th2-biasing CD1d glycolipid ligands that have modification at their lipid part of α-GalCer structure. The modifications lead to high binding affinities and efficient Th2 cytokine production. The appearance rates of ligand-CD1d complexes on the cell surface were also involved in Th2-biasing responses. A covalent α-GalCer derivative was also developed as potent CD1d ligands, which showed Th2-biasing response. We here demonstrate the importance of lipid binding affinity of glycolipids to the receptor proteins in the lipid antigen presentation, for the modulation of cytokine-biasing responses.
Tuesday

Tuesday
Glycosylations represent one of the most mechanistically complex organic transformations, while stereoselectivity is one of the more nuanced and sensitive outcomes of a chemical transformation. In this talk, I will describe how a random forest algortihm, trained on a concise dataset generated on a continuous flow platform, is able to accurately predict the α/β-selectivity of a range of glycosylation reactions, varying donor, acceptor, solvent, activator, and temperature.1,2 These predictions were all validated experimentally on the same flow platform. The output of the model also provides insight into degree of influence of each of the eleven independent variables influencing stereoselectivity, such as the significant selectivity preferences locked in once the coupling partners are chosen. Further, the model accurately predicts previously undocumented means of influence over glycosylation stereselectivity.
Tuesday
Uropathogenic E. coli (UPEC) initiates urinary tract infections (UTIs) via the interaction of lectin FimH at the tip of E. coli pili binding to oligo-alpha-mannosides on urothelial glycoprotein uroplakin 1a. A strategy for the treatment of UTIs is the anti-adhesin approach, where a soluble ligand acts as an antagonist by competing with the native glycoprotein. The antagonists targeting this interaction are mostly alpha-linked mannose glycosides that have various aglycone moieties. We recently reported on a 1-deoxy-2-O-n-heptlyl septanoside as a flexible analog of n-heptyl alpha-mannoside that bound to the FimH lectin domain. Using thermodynamic and structural information from that study, new septanose ligands were designed, synthesized, and evaluated for binding to FimH. The synthesis leverages a key oxepine intermediate to allow for placement of the C2 hydroxyl group. Varying aryl groups have been added to the C2, allowing for a rapid generation of ligands. This is done using a base-mediated Sn2 alkylation followed by a Suzuki coupling with varying phenylboronic acids. These aryl groups were selected based on mannoside aglycones that have been shown in literature to give strong binding to FimH. Comparing these septanose ligands to their previously tested pyranose counterparts will allow for evaluation of the different rings with binding pocket size and carbohydrate flexibility factors in the difference of binding affinity. Insights gained can be used in expanding outward and applying septanoses as antagonists in other proteins, increasing their therapeutic potential.
Tuesday
Diabetes mellitus is a severe, chronic disease that affects over 420 million people worldwide. If left untreated, diabetes can lead to severe complications and death. The onset of complications can be delayed or even prevented when the disease is diagnosed early, but it is estimated that nearly 40% of all persons with diabetes are undiagnosed. Screening in communities with limited access to healthcare can significantly reduce the number of undiagnosed cases of diabetes. The blood concentration of 1,5-anhydroglucitol (AHG), a naturally-occurring six-carbon monosaccharide similar in structure to glucose, falls during periods of hyperglycemia as glucose outcompetes AHG for kidney reuptake. Blood AHG measurement is an FDA-cleared method of monitoring glycemic control, and salivary AHG has been suggested to be useful for diabetes screening. However, previous efforts to measure AHG in saliva have been unsuccessful. We have developed a chemiluminescence assay to quantify AHG in saliva and demonstrated that the assay could distinguish between healthy and treated-diabetic individuals (N=265; p < 0.001, ROC AUC 0.82). These results suggest that, with further validation, this approach may serve as the basis of a non-invasive tool to screen for diabetes.
Division/Committee: [BIOL] Division of Biological Chemistry

This symposium will honor women scientists working in the field of bioconjugate chemistry, sponsored by the journal Bioconjugate Chemistry.
Wednesday

Wednesday
The blockade of programed death-1 receptor (PD-1) or its ligand PD-L1 has been a breakthrough in cancer therapies. However, the objective response rate of PD1/PD-L1 blockade monotherapy in various cancers is only 20-30%. Therefore, combination therapy has become the current immunotherapy strategy, especially in combination with radiation therapy and chemotherapy, and more than 1,700 clinical trials of combination therapy are currently underway worldwide. In clinical trials, how to enhance the effectiveness of combination therapies and reduce toxicity is always the focus of attention.
We reported an anti-PD-L1 (αPD-L1) nanobody probe (99mTc-MY1523) for single photon emission computed tomography (SPECT) imaging to non-invasively evaluate the expression of PD-L1 in tumors, and proposed the concept of time window of αPD-L1 immumotherapy, that is, when the PD-L1 expression in tumors was high or upregulated as determined by SPECT could be a favorable time window for initiation of immunotherapies. The results revealed that the imaging-guided αPD-L1 immunotherapy significantly enhanced the therapeutic effect.
We fabricated a novel albumin-binding (AB) entity conjugated long-acting RGD analogue 177Lu-AB-3PRGD2 with improved tumor uptake and tumor residence than 177Lu-3PRGD2 for integrin αvβ3-targeted radionuclide therapy. The results validated that the low dose of 177Lu-AB-3PRGD2 could upregulated the PD-L1 expression in tumors, and imaging-guided combination of 177Lu-AB-3PRGD2 radiotherapy and αPD-L1 immunotherapy synergistically enhanced the antitumor efficacy.
(A) Representative SPECT/CT images of PD-L1 expression in tumors with 99mTc-MY1523 after the low dose 177Lu-AB-3PRGD2 treatment for 3, 6, 9 and 12 days (arrows indicate the tumors). Synergistic antitumor efficacy in the MC38 tumor-bearing mice. (B) αPD-L1 monotreatment (100 µg, i.v.) on day -3, 0 and 3 (n = 7, mean ± SD). (C) Combination therapy of 100 µg αPD-L1 (on day -3, 0 and 3) and 9 MBq 177Lu-AB-3PRGD2 (on day 0) (n = 7, mean ± SD). (D) Mice survivals after combination therapy (n = 7, mean ± SD).

(A) Representative SPECT/CT images of PD-L1 expression in tumors with 99mTc-MY1523 after the low dose 177Lu-AB-3PRGD2 treatment for 3, 6, 9 and 12 days (arrows indicate the tumors). Synergistic antitumor efficacy in the MC38 tumor-bearing mice. (B) αPD-L1 monotreatment (100 µg, i.v.) on day -3, 0 and 3 (n = 7, mean ± SD). (C) Combination therapy of 100 µg αPD-L1 (on day -3, 0 and 3) and 9 MBq 177Lu-AB-3PRGD2 (on day 0) (n = 7, mean ± SD). (D) Mice survivals after combination therapy (n = 7, mean ± SD).


Wednesday
Metastasis is the leading cause of fatality for women diagnosed with breast cancer. The most common anatomical sites of distant tumor growth include the brain, lung, liver, and bone, and it is well known that this metastatic spread in breast cancer is not random. Rather, different subtypes of breast cancer exhibit unique patterns of metastatic site preference. Given the physical and chemical diversity of these secondary tissue sites, my lab hypothesizes that there is a relationship between the biophysical and biochemical properties of the tissue, and the ability of cells within a particular subtype of breast cancer to adhere, migrate, grow, and respond to chemotherapy at these secondary sites. We create biomaterial microenvironments, which capture some of the key physical and biochemical elements of these tissues (brain, lung, and bone). Our approach is revealing how cell-material interactions are predictive of where cancer spreads, how some cancer cells can be ‘put to sleep’ at these distant sites, and how we can use rational material design to identify new, more powerful drug regimens to kill cancer cells more efficiently. I will discuss our lab’s emphasis on zwitterion-containing hydrogels to achieve these efforts, which we propose can predict metastasis in patients, and may serve as better systems to identify patient-specific cancer treatments.
Wednesday
Cell-based brain repair is a promising option for Parkinson’s disease (PD) whereby the nigrostriatal dopaminergic neurons that have degenerated over the course of the disease are replaced by transplantation of healthy neurons into the brain. Given that cell-based brain repair is rapidly accelerating towards the clinic, with the ongoing TRANSEURO trial of fetal tissue and the recently announced Takahashi trial of iPSC-derived dopaminergic neurons in Japan, but that the margin for improvement of such approaches is great, it is critical to continue rigorous preclinical studies to identify potential methods of improving the outcome of cell-based brain repair for patients.

In this context, we will be presenting our recent data demonstrating that dopaminergic cell replacement in the Parkinsonian rodent brain, using fetal-derived cells, is dramatically enhanced when the cells were transplanted in a neurotrophin-enriched, immune-shielding collagen hydrogel. The hydrogel provided the transplanted neurons with 1) a physical scaffold for cell-matrix adhesion, 2) a neurotrophin reservoir for sustained neurotrophin exposure after transplantation, and 3) shielding from the deleterious effects of the host microglial and astroglial innate immune response (Fig. 1). We will also present data from studies encapsulating human iPSC-derived dopaminergic neurons.

Overall, this work suggests that the clinical transplant field should move towards the incorporation of biomaterials, such as neurotrophin-enriched collagen hydrogels, into future clinical trials using primary and/or iPSC derived neurons. Improving the safety and efficacy of such approaches, using this minimally invasive and injectable hydrogel that offers a neuroprotective and immune shielding microenvironment to the transplanted cells, could dramatically improve the reparative capacity of cell therapy for PD, and ultimately lead to an improved therapy for patients.
<b>Fig. 1 Schematic illustrating some of the benefits of neurotrophin-enriched injectable collagen hydrogels for cell-based brain repair. </b>

Fig. 1 Schematic illustrating some of the benefits of neurotrophin-enriched injectable collagen hydrogels for cell-based brain repair.


Wednesday
Recent developments in photodegradable hydrogels have allowed researchers to study cell behavior in response to spatial and temporal changes to the extracellular environment. To date, most photodegradable hydrogel systems have been composed of poly (ethylene glycol) (PEG) based macromers that crosslink via end-linking gelation. PEG-based hydrogels, however, are not optimal for three-dimension cell culture, as they neither allow for cellular proliferation nor restructuring of the matrix. Unlike PEG-based hydrogels, gelatin, a naturally derived material, contains enzymatically degradable sites and cell binding domains, making it an attractive biomaterial for three-dimensional cell culture. Gelatin is typically modified with methacrylamide groups to allow for crosslinking, which enhances and enhances its stability under physiological conditions. A few groups have reported the synthesis of photodegradable gelatin, but the incorporation of photodegradable groups is hampered by poor conjugation efficiency and poor solubility, leading to insufficient mechanical properties. We report a photodegradable gelatin hydrogel system that is mechanically robust and can be easily produced in large quantities. Specifically, we chemically modify the gelatin with highly hydrophilic groups which, in turn, adjust the isoelectric point and charge density of the protein. This modification results in a highly soluble photodegradable -gelatin that can be crosslinked into a gel and subsequently degraded with exposure to light. These photodegradable -gelatin gels exhibit mechanical properties similar to gelatin methacrylamide gels, but with the extra ability to be spatially and temporally patterned with light. Photodegradation of the gels can be done either before cell seeding or in the presence of cells. We show that cells respond to both patterned structures and in situ softening of the network in 2D, while in situ softening in 3D does not affect morphology at the compositions and time scales investigated.
Wednesday

Wednesday
Cationic polymers are versatile alternatives to engineered viruses for the delivery of genome editing payloads. Yet, their clinical translation hinges on facile exploration of broad chemical design spaces and deriving structure-function relationships governing performance. We have discovered a polymer for efficient intracellular CRISPR-Cas 9 ribonucleoprotein (RNP) delivery through combinatorial polymer design and parallelized experimental. A chemically diverse library of 43 statistical copolymers has been synthesized via RAFT polymerization, realizing systematic variations in physicochemical properties. This multiparametric polymer library was examined througha variety of physical and biological characterization techniques, which rapidly uncoveredahitpolymer that outperformed commercial transfection reagents, achieving nearly 60% editing efficiency via non-homologous end-joining. Structure-function correlations underlying editing efficiency, cellular toxicity, and RNP uptake were probed through machine learning approaches to uncover the physicochemical basis of delivery performance. Polymer hydrophobicity and the Hill coefficient, a parameter describing cooperativity-enhanced polymer deprotonation, were identified as the critical determinants of editing efficiency. Combinatorial synthesis and high-throughput characterization methodologies coupled with data science approaches enabled the rapid discovery of a novel polymeric vehicle that would have otherwise remained inaccessible to chemical intuition.

Wednesday
Several diseases are attributed to either deficient or excessive vascularization, so understanding angiogenesis offers great promise for improving vascular therapies. Angiogenesis is primarily regulated by vascular endothelial growth factor receptors (VEGFRs), and changes in VEGFR protein expression are promising biomarkers for predicting the efficacy of VEGFR pathway inhibition. However, current data on VEGFR protein expression in human vessel biopsies are limited, given the invasive procedure necessary and difficulty in standardizing non-quantitative measurements across labs. Here, we pioneer a standardized workflow that consists of liquid biopsy-based quantitative flow cytometry and cell-by-cell data analysis. We employed this workflow to establish healthy baselines for VEGFRs on the plasma membranes of two key circulating angiogenic cell populations: circulating endothelial cells (cECs) and circulating progenitor cells (cPCs). Our findings provide new insights into how sex and age influence the distribution of VEGFRs on these circulating angiogenic cells.
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Rotator cuff injuries represent a range of pathologies, from early tendon overuse to full thickness rotator cuff tendon tears. Once the tendon has undergone full thickness tear, marked degeneration of the attached muscle is apparent clinically, with both fibrous and fatty infiltration of the tissue. Our laboratory is working on delivery strategies for biologics, including proteins and cells, that might slow or reverse this degeneration. In particular, in this presentation, we focus on “jump-starting” host regenerative processes through use of glycosaminoglycan (GAG)-based biomaterials for release of cytokines to recruit pro-healing cell populations. In other work, we explore direct transplantation of culture-primed mesenchymal stem cells (MSCs) as a strategy for reducing muscle degeneration after rotator cuff tendon tear.
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Division/Committee: [CARB] Division of Carbohydrate Chemistry

This Symposium will encompass recent developments in all areas of
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Inverse electron-demand [4+2] cycloadditions involving glycals as electron rich dienophiles
and alpha,alpha’-dioxothiones as electron poor dienes, are powerful reactions to afford 2-deoxy-2-thio-O-glycosides chemo-, regio- and stereoselectively.
Relying on these versatile reactions, diastereomerically pure O-glycosides have been prepared and employed as starting material to obtain a collection of oligosaccharides, glycopeptides and glycoproteins.
Thanks to the structure versatility, selected thio-glycosides have been used to prepare bio-inspired glyco-materials decorating multivalent constructs, including nanoparticles, nano fibers, a cyclopeptidic (RAFT) scaffold as well as proteins.
Binding studies and biological assays performed with some of these multivalent architectures will be presented.
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Carbohydrates are essential biomolecules made by every living organism. Our cells are coated with sugars that are involved in almost every biological process and defensive mechanism in our body. Carbohydrates participate in blood coagulation, immune defense, cell growth, cell-cell interaction and anti-inflammatory processes. Thus, understanding of glycan function and structure is crucial for the development of vaccines and theranostics. This also makes carbohydrates important synthetic targets in the modern medicinal chemistry field.

Thioglycosides are one of the most popular building blocks used both for modification of monosaccharides and assembly of glycans. Since their first activation in 1973, a great variety of promoters for thioglycoside activation have been developed. Previously, our lab reported 3,3-difluoroxindole (HOFox) based regenerative glycosylation reaction. OFox are very reactive O-imidoyl leaving groups, and the regenerative approach comprises both the in situ synthesis and activation of OFox glycosyl donors in a catalytic regenerative fashion. The original regenerative reaction involved transformation of thioglycoside precursors into glycosyl bromides. The latter was then activated in the presence of Ag2O and HOFox, to introduce the OFox leaving group. The latter is then activated with a catalytic Lewis acid thereby generating HOFox that becomes available to continue glycosylation in a regenerative fashion. Herein we report the development of a streamlined approach for the direct glycosidation of thioglycosides that would bypass the intermediacy of bromides and eliminate the need for heavy metal-based stoichiometric promoters.
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Despite substantial progresses in the prevention of group B Streptococcus (GBS) disease with the introduction of intrapartum antibiotic prophylaxis, this pathogen is a leading cause of neonatal infections. On the basis of variation in the sugar composition of the capsular polysaccharide (CPS), which is a major virulence factor, ten serotypes of GBS have been identified: CPS conjugate vaccines representing several serotypes have been tested in phase I/II clinical studies, showing promise for further development. The elucidation of polysaccharide epitopes is relevant for understanding the mechanism of action of glycoconjugates and designing synthetic carbohydrate-based vaccines. Recently, an X-ray/NMR based approach was applied to determine a functional epitope of GBS PSIII, which resulted composed of six residues included within two repeating units, paving the way towards the use of synthetic structures for vaccine development.[1]
The repeating units of GBS serotypes Ia, Ib and III share similarities in their sugar composition, such as the NeuNAcβ(1–>3)Gal branch and the GlcNAcβ(1–>3)Gal motif. We envisaged the regioselective glycosylation of galactose 3-OH to form the disaccharide motif GlcNAcβ(1–>3)Gal as a key step for the synthesis of fragments of the three serotypes. A synthetic approach taking advantage of the well-recognized major reactivity of Gal 3-OH compared to the 4-OH was designed: a number of glucosamine donors and galactose acceptors were screened to optimize the regioselective glycosylation for faster access to the target disaccharide.
With this synthetic design the pentasaccharide repeating unit of GBS CPS serotype Ib was synthesized[2], as well as oligosaccharides from serotype Ia and the GBS type III hexasaccharide corresponding to the structural minimal epitope.[3] The oligosaccharides were screened with a combined approach, including competitive SPR and STD-NMR, to investigate their interactions with mAbs and to identify optimal or sub-optimal glycotopes for conjugation to carrier proteins and immunological evaluation.
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Chemotherapy and host immunity both impose stresses on bacterial pathogens resulting in protein aggregation. Eukaryotic Hsp104/Hsp70 and their bacterial homologs ClpB/DnaK are ATP-powered chaperones that restore toxic protein aggregates to a native folded state. DnaK is predicted to be essential in several species of mycobacteria, including the pathogen M. tuberculosis (Mtb), which is the infective agent of tuberculosis (TB). We and others have shown that ClpB mediates a stress response to sublethal doses of antibiotic in Mtb. Both DnaK and ClpB are potential drug targets in mycobacteria; however, their molecular partners in protein reactivation and modes of inhibition have not been thoroughly explored. We have performed small molecule high-throughput screening against mycobacterial DnaK and known cofactors, and have identified small molecule inhibitors that target allosteric sites on DnaK. In this talk, we characterize the modes of binding of these small molecules and discuss strategies to selectively target bacterial chaperones based on their mechanisms of inhibition.
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Toll-Like Receptor 4 (TLR4) is one of the receptors of innate immunity, it is activated by Pathogen- and Damage-Associated Molecular Patterns (PAMPs and DAMPs), and triggers pro-inflammatory responses which belong to the repertoire of innate immune responses, thus protecting against infectious challenges and boosting adaptive immunity. Mild TLR4 stimulation by non-toxic molecules resembling its natural agonist (lipid A), provided efficient vaccine adjuvants. The non-toxic TLR4 agonist monophosphoryl lipid A (MPLA) has been approved for clinical use, thus suggesting the development of other TLR4 agonists as adjuvants or drugs for cancer immunotherapy. TLR4 excessive activation by Gram-negative bacteria lipopolysaccharide (LPS) leads to sepsis, while TLR4 stimulation by DAMPs is a common mechanism in several inflammatory and autoimmune diseases. TLR4 inhibition by small molecules and antibodies could therefore provide access to innovative therapeutics targeting sepsis, acute and chronic inflammations. TLR4 antagonists can also block the violent inflammation and cytokine storm caused by several viral diseases, including COVID-19.
The most recent achievement of our group in the development of synthetic glycolipids with activity as TLR4 agonists and antagonists (FP molecules) will be presented: computer-assisted rational design, in vitro binding studies with the receptor, cell and in vivo activity, studies on the mechanism of action.
New TLR4 agonists with a monosaccharide structure mimic the pharmacophore of lipid A and MPLA, and potently stimulate innate immunity and are in preclinical development as adjuvants in antiviral and antibacterial vaccines (www.bactivax.eu). Monosaccharide antagonist FP7 efficiently blocked inflammation in cell models and in animal models of sepsis, influenza virus lethality, vascular inflammations, neuroinflammations, and inflammatory bowel diseases (IBDs).

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Infective endocarditis (IE) is a cardiovascular disease caused by the entry of oral bacteria in the blood stream and the subsequent colonization of microbes in platelet–fibrin thrombi on cardiac valve surfaces. Streptococcus gordonii and Streptococcus sanguinis, commensal species among the normal oral microbiota, become opportunistic pathogens that can cause IE.
The presence of "Siglec-like" serine-rich repeat adhesins on the microbial surface may increase the propensity of streptococci to cause IE. These adhesins contain Siglec-like binding regions (SLBRs) that recognize α2-3 sialylated glycan structures, including O-linked glycans displayed on salivary MUC7, platelet GPIb and several mucin-like plasma proteins. The SLBRs from different strains recognize different repertoires of structures, with some displaying selectivity for a single structure and others showing broader specificity. GspB and Hsa are the Siglec-like serine-rich repeat adhesins of S. gordoni strains M99 and Challis, respectively, that have the ability to mediate Streptococcus adhesion to platelet membrane glycoproteins. The high-resolution crystal structures of these proteins have been published, but they have not fully explained the determinants of ligand specificity.
Thus, unveiling the mechanism of glycan recognition by Siglec-like adhesins, at molecular level, represent a key step to better understand the different selectivity and flexibility of the streptococcal adhesins towards sialoglycans.
In this context, we explored the recognition and binding of sialoglycans by different siglec-like adhesins, including Hsa and GspB. The results provided a detailed description of the conformational behavior of sialoglycans upon binding and allow to achieve a 3D view of the protein-ligand complexes. These outcomes were obtained by using several techniques, in particular NMR ligand-based methods, such as Saturation Transfer Difference NMR and transferred NOESY, as well as computational approaches, including docking, Molecular Dynamics and CORCEMA-ST analysis.
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Pneumonia is a serious respiratory infection mainly caused by Streptococcus pneumoniae (SP) bacterium. According to a recent report, one child every 39 seconds dies of invasive pneumococcal disease (IPD). Since the polysaccharide capsules of SP are key virulence factors, commercially available vaccines rely on pneumococcal capsular polysaccharide (CPS) antigens able to induce serotype-specific immune responses. Pneumococcal conjugate vaccines (PCVs) have substantially reduced the incidence of IPD caused by the most virulent serotypes included in vaccine formulations. However, newly reported IPD cases are mainly caused by emergent serotypes not included in PCV formulations, addressing the continuous need of increased multivalency for a broadened coverage.
Since we are far from reaching a not serotype-dependent vaccine, we envisioned that PCV formulations can be simplified by designing novel saccharide antigens, potentially able to induce an immune response targeting more than one serotype. In this context, SP 19F and 19A CPS repeating units share a common structure, the disaccharide ManNAc-β-(1→4)-Glc. We have synthesized a set of compounds characterized by different combinations of this common disaccharide, and evaluated their ability to bind anti-19F and anti-19A antibodies by glycan array analysis.

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A hallmark of all cancers is the aberrant expression of cell-surface glycans; these aberrations cause distinct changes in cellular properties such as adhesion, signaling and metastasis. Many of these modified structures are known as Tumor-Associated Carbohydrate Antigens (TACAs) for their ability to elicit immune responses, and circulating antibodies to TACAs can be protective. One TACA disaccharide, the Thomsen-Friedenreich antigen (TF-ag), has been the target of a host of antitumor vaccine designs, while also being a motif that is directly linked to the metastatic spread of various solid tumor. We have developed gold nanoparticle platforms bearing the TF-ag or TF-ag-containing glycopeptides as both antimetastatic agents or immunotherapeutics. The design and development of these platforms along with their biological relevance will be discussed. In addition, we use various techniques to define the structural basis for the function of these constructs.
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