CARB 1

Building on Wolfrom’s legacy: From the Chemurgy of yesterday to the renewables of today

Kevin B. Hicks1, kevin.hicks@ars.usda.gov, Akwasi A. Boateng4, Charles A. Mullen5, Yaseen Elkasabi1, Arland T. Hotchkiss2, Madhav P. Yadav3. (1) Eastern Regional Research Center, ARS, USDA, Malvern, Pennsylvania, United States (2) Errc, USDA ARS, Wyndmoor, Pennsylvania, United States (3) SBCP, USDA, ARS, ERRC, Wyndmoor, Pennsylvania, United States (4) Eastern Regional Research Center, ARS, USDA, Wyndmoor, Pennsylvania, United States

The early part of the 20th century gave rise to the “Chemurgy” movement in which researchers worked to produce valuable industrial chemicals, fuels, and plastics from surplus agricultural crops and forestry products. That movement lost momentum as the petroleum refining industry rapidly advanced and began to economically produce practically any chemical desired. Concerns about climate change, energy security, and oil reserves helped spawn a new interest in research on biofuels and other renewables during the end of the 20th century and the beginning of the 21st. But recent advances in hydraulic fracturing and the resulting cheaper petroleum and natural gas threatens to derail the current interest in renewables and biofuels once again. Regardless of the present outcome, we will ultimately need to turn to renewable sources of feedstocks for production of all our needed foods, feeds, fuels, and chemicals. In this grand endeavor, carbohydrates and carbohydrate chemists will play key roles. Two emerging and promising routes to convert biomass to fuels and chemicals will be discussed. First considered will be how the production of high-valued polysaccharide coproducts may help increase the economic viability of the pioneering cellulosic ethanol facilities started up in 2014/2015. Next to be discussed will be a new thermochemical process called Tail Gas Reactive Pyrolysis, which has the potential to convert non-food biomass into drop-in fuels and chemicals. The successful implementation of new technologies such as these can help renewable fuels and chemicals compete more effectively with fossil-derived products in the future.

CARB 2

Cranberries: From polyphenols to polysaccharides

Christina Khoo, ckhoo@oceanspray.com. Research Sciences, Ocean Spray Cranberries, Inc, Lakeville, Massachusetts, United States

The North American cranberry, Vaccinium macrocarpon, has one of the highest polyphenol content of commonly consumed fruits. Due to the rich polyphenol and naturally low sugar content, cranberries are also tart and astringent, requiring sweetener for palatability. Cranberries are rarely consumed raw so most studies were conducted with cranberry juice and powders. Characterization of cranberry compounds in these products is important to understand the well- known health effects including urinary

tract, heart and gastrointestinal health. These benefits are thought to be conferred by bioactive flavonoids such as flavan-3-ol, anthocyanins, flavonols and polymeric proanthocyanidins; the Type A and Type B proanthocyanidins. Polyphenols are now thought to have activity beyond suppressing free radicals and instead, are thought to help modulate our body’s antioxidant defense system and reduce oxidative stress. The health promoting properties of cranberries linked to urinary tract health are thought to be due to the activity of proanthocyanidins, especially type A proanthocyanidins (PACs). The mechanism by which cranberries are efficacious in preventing UTIs has yielded many hypotheses, including preventing bacterial adhesion in the bladder and gut, immune modulation and effects on bacterial fimbriae expression. Therefore, there is active ongoing research exploring the bioavailability and activity of cranberry metabolites as well as metabolites of gut flora metabolism to understand their contribution to the benefits attributed to cranberries. Recent publications also provide evidence that phenolic acids abundant in cranberry are present after cranberry juice consumption and may help confer putative health benefits. In addition, a new and exciting avenue of research is now focused on cranberry oligosaccharides, recently characterized and identified. More research is needed to understand the contribution of these compounds and their synergies in support of cranberry’s health benefits.

CARB 3

Bioactive pectic oligosaccharides

Arland T. Hotchkiss, arland.hotchkiss@ars.usda.gov. Agricultural Research Service, US Department of Agriculture , Wyndmoor, Pennsylvania, United States

For 12 years, evidence has accumulated for the prebiotic properties of pectic oligosaccharides (POS). Data is based on fluorescence in situ hybridization using 16s rRNA probes of fecal fermentations supplemented with oligosaccharides. Several methods have been used to produce POS fractions with bifidogenic and other prebiotic properties. Structure-function studies have demonstrated that the arabino- and galacto-oligosaccharide pectin side chains are responsible for prebiotic properties in mixed batch fecal fermentations. Homogalacturonan oligosaccharides are active in blocking the adhesion and invasion of food-borne pathogens. Pectic galacto-oligosaccharides and modified citrus pectin antagonize galectin-3 mediated tumor cell metastasis and have immunomodulatory activity. Preliminary POS animal data will be presented and POS infant formula human clinical data will be reviewed. The composition of pectic oligosaccharides influences T helper cell immune responses and inflammation. Therefore, POS is very important to understanding how the gut microbiome impacts our health and the control of chronic diseases.

CARB 4

Recent studies on the synthesis of glycans from mycobacteria and campylobacters

Todd L. Lowary, tlowary@ualberta.ca. Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada

Mycobacteria and campylobacters produce a range of structurally diverse glycans with a host of biological activities. In this seminar recent work from the group on the synthesis of these molecules will be presented. Particular emphasis will be on lipooligosaccharide antigens from mycobacteria and methyl phosphoramidate (MeOPN)-containing glycans from Campylobacter jejuni.

CARB 5

Synthesis of heparan sulfate oligosaccharides and glycopeptide

Xuefei Huang2, xuefei@chemistry.msu.edu, Steven B. Dulaney2, Yongmei Xu1, Keisuke Yoshida2, Bo Yang2, Weizhun Yang2, Jian Liu1. (1) Eshelman Schl of Pharmacy, University of North Carolina, Chapel Hill, North Carolina, United States (2) Chemistry, Michigan State University, East Lansing, Michigan, United States

Heparan sulfates are highly sulfated polysaccharides exhibiting important functions in many biological processes. The backbone structures and sulfation patterns determine their biological activities. In nature, heparan sulfates exist as part of heparan sulfate proteoglycans with the glycan chain linked to the core protein backbone through a tetrasaccharide linker. Despite the ubiquitous presence of proteoglycans in mammalian systems, methodologies to synthesize this class of glycopeptides bearing homogenous glycans are not well developed. In this presentation, we will discuss our recent results on synthesis of heparan sulfate oligosaccharides and glycopeptides through a combination of chemical and enzymatic methods. Advanced intermediate oligosaccharides were prepared by chemical synthesis, which were subjected to enzymatic modifications. Through this approach, we have obtained multiple structurally defined heparan sulfate oligosaccharides with designed biological activities. Our efforts in extending this methodology towards the preparation of heparan sulfate glycopeptides will also be presented.

CARB 6

Therapeutic in vivo synthesis by glycocarriers

Katsunori Tanaka, ktzenori@chem.sci.osaka-u.ac.jp. Biofunctional Synthetic Chemistry Laboratory, RIKEN, Wako, Saitama, Japan

Through a concept we refer to as "Therapeutic In Vivo Synthetic Chemistry", we aim to develop an adaptable system where a cascade of organic transformations can be directly executed at target regions within the body during predefined times to generate a bioactive molecule that elicits a localized biological effect. Towards this goal, we are analyzing, with the use of molecular imaging, the complex pattern recognition mechanisms of natural glycans in vivo and applying the glycan-based interactions to

direct various linked biomolecules to desired organs and tissues. Our research has introduced various glycan structures onto proteins, dendrons, and live cells, through the 6π-azaelectrocyclization protocols [1-4], as a means to investigate glycan dependence on in vivo dynamics. Through the use of molecular imaging, results have shown that glycan composition on these templates can directly control accumulation towards specific organ and cancer cells, as well as affect their excretion profiles. We are ultimately challenging to develop a model system where bioactive compounds can be synthesized within living animals, i.e., "Therapeutic In Vivo Synthetic Chemistry". Our preliminary challenge will be discussed in this symposium. [1] K. Tanaka, T. Masuyama, K. Hasegawa, T. Tahara, H. Mizuma, Y. Wada, Y. Watanabe, K. Fukase, Angew. Chem. Int. Ed., 2008, 47, 102-105. [2] K. Tanaka, E. R. O. Siwu, K. Minami, K. Hasegawa, S. Nozaki, Y. Kanayama, K. Koyama, C. W. Chen, J. C. Paulson, Y. Watanabe, K. Fukase, Angew. Chem. Int. Ed., 2010, 49, 8195. [3] K. Fukase, K. Tanaka, Curr. Opin. Chem. Biol., 2012, 16, 614-621. [4] A. Ogura, K. Tanaka, to be submitted.

CARB 7

Split personality of human O-GlcNAc transferase

Suzanne Walker, suzanne_walker@hms.harvard.edu. Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, United States

O-GlcNAc Transferase (OGT) is an essential glycosyltransferase that catalyzes the attachment of N-acetylglucosamine (GlcNAc) to serines and threonines of numerous nuclear and cytoplasmic proteins. Global O-GlcNAcylation is sensitive to changes in the concentration of OGT’s substrate, UDP-GlcNAc, which is affected by cellular glucose levels. Increased O-GlcNAcylation is associated with widespread changes in gene

expression, but the molecular mechanisms underlying these changes remain poorly understood. Recently, OGT was shown to catalyze another post-translational modification: the cleavage of the cell cycle regulator Host Cell Factor 1 (HCF-1). We will describe our work on the unusual mechanism of HCF-1 cleavage by OGT. We will show that cleavage requires UDP-GlcNAc as a cosubstrate, implying that HCF-1 cleavage, like O-GlcNAcylation, is coupled to UDP-GlcNAc concentration. Hence, key cell cycle regulatory functions of HCF-1 are linked to nutrient levels through a remarkable proteolytic action of OGT.

CARB 8

Photocrosslinking approach to discover O-GlcNAc-interacting proteins

Andrea Rodriguez, Seok-Ho Yu, Bin Li, Jennifer J. Kohler, Jennifer.Kohler@UTSouthwestern.edu. University of Texas Southwestern Medical Center, Dallas, Texas, United States

O-GlcNAc is an abundant and reversible modification of mammalian proteins. While hundreds of O-GlcNAc-modified proteins have been identified, relatively little is known about how O-GlcNAc affects protein function. Intriguingly, recent reports suggest that one functional role for O-GlcNAc is to promote protein-protein interactions. This talk will describe development of two photocrosslinking methods to discover O-GlcNAc-dependent interaction partners. The first method takes place in living cells and relies on mutagenesis of two mammalian enzymes (UAP1 and OGT) to achieve metabolic incorporation of a diazirine-modified GlcNAc analog (O-GlcNDAz). The second method is an in vitro approach that depends on chemoenzymatic production of diazirine-modified O-GlcNAc-ylated peptides. Implementation of the in vitro approach requires mutagenesis of one bacterial enzyme (NahK) and two mammalian enzymes (OGT and OGA) to facilitate conversion of diazirine-modified substrates. Both approaches are being used to identify the interaction partners of O-GlcNAc-modified nucleoporins and to assess how these interactions change in response to stress and other perturbations.

CARB 9

Chemical probes for the functional analysis of O-GlcNAc modifications

Matthew Pratt, matthew.pratt@usc.edu. University of Southern California, Los Angeles, California, United States

The modification of proteins in the cytosol, nucleus, and mitochondria by the monosaccharide N-acetyl-glucosamine (O-GlcNAc) is required for development in mammals and Drosophila and is misregulated in a variety of diseases including cancer. One of the general functions of O-GlcNAcylation is to respond to changes in metabolism and cellular stress. Here I will present the use of both chemoenzymatic labeling and new metabolic chemical reporters to investigate how O-GlcNAcylation levels become increased during oxidative stress and how this increase promotes cell-survival.

CARB 10

Hexosamine mimetics designed to reverse the Warburg Effect

Kevin J. Yarema, kyarema1@jhu.edu. The Johns Hopkins University, Woodstock, Maryland, United States

The Warburg Effect refers to an almost century old, but newly relevant, ubiquitous feature of cancer wherein cells ferment large amount of glucose while largely forgoing oxidative phosphorylation. As a consequence of this aberrantly high glucose intake, flux through the hexosamine biosynthetic pathway (HBP) increases dramatically in cancer cells leading to production of various types of glycans that exacerbate cancer progression. This presentation will provide an overview of three classes of hexomine-based glycomimetics designed to either directly or indirectly reverse the adverse impacts of the Warburg Effect that emanate from enhanced flux through the HBP. The first category targets UAP1/2, the final enzyme in the HBP before production of the critical metabolite UDP-GlcNAc. The second category is comprised of ManNAc analogs that dramatically increase sialylation, an effect normally associated with cancer progression, but in this case provides sensitization to extant cancer drugs. Finally, a third category gains anti-cancer activity from the specific arrangement of esterase-linked n-butyrate groups on the core hexosamine moiety.

CARB 11

Selectin antagonists: Acyclic tethers with a define conformational bias

Yvan Guindon, yvan.guindon@ircm.qc.ca, Calosso Mickael, Guillaume Tambutet, Michel Prévost. Institut de Recherches Cliniques de Montreal, Montreal, Quebec, Canada

Sialyl Lewisx is one of the natural ligands of E- and P-selectins. Our group showed that Glc-NAc, a polysaccharide subunit that holds in space the fucose and galactose derivative of Sialyl Lewisx, could be advantageously replaced by acyclic tethers possessing a defined conformational bias to provide high levels of E- and P-selectin

blocking activity, both in vitro and in vivo. The design and stereoselective synthesis of these molecules, and other analogues thereof, will be presented.

CARB 12

Toward the development of selective DC-SIGN antagonists

Anna Bernardi, anna.bernardi@unimi.it. Dept. of Chemistry, Universita degli Studi di Milano, Milano, Italy

Over the past few years, our group has been designing and synthesizing glycomimetic antagonists of mannose-specific C-lectins. The design takes advantage of the 3D structure of known oligosaccharide ligands and of available structural information on the lectin/ligand complexes. The small-molecule monovalent ligands obtained are often endowed with limited protein affinity, but display improved drug-like properties compared to natural sugars. Multivalent presentation on polymeric scaffolds has afforded high-affinity antagonists, and the selectivity of these materials against different C-lectins is being investigated. DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule 3-Grabbing Nonintegrin), a specific C-type lectin that recognizes highly mannosylated proteins on pathogen surfaces, is a trans-membrane receptor of immature dendritic cells (DC) which binds a number of pathogen-associated molecular patterns. Normally, this binding event triggers internalization of the DC-SIGN-pathogen complex, followed by lysosomal degradation and conjugation of the resulting fragments with MHC-II to initiate an adaptive immune response from T cells. However, some pathogens, including viruses like HIV, Dengue and Ebola, have been reported to highjack this mechanism, as they appear to deter DC maturation through DC-SIGN-mediated signalling and inhibit antigen presentation to T cells. By doing so these viruses effectively exploit DC-SIGN to fully disseminate the infection. Inhibition of pathogen interaction using DC-SIGN specific antagonists is considered a plausible concept for the development of novel anti-infective agents. Several groups

have recently demonstrated that inhibition of DC-SIGN, either by designed glycoconjugates or by antibodies, prevents pathogen attachment to DC and inhibits the infection of other immune cells at its earliest steps. Our group has been active in this area and, in collaboration with the European Network Carmusys, has developed mannose-based and fucose-based glycomimetic ligands that inhibit DC-SIGN mediated viral infection in cellular and tissue models. The presentation will deal with the design and synthesis of the glycomimetic monovalent ligands, the process of structural optimization which allowed us to build affinity and selectivity in their structure, the synthesis and optimization of polyvalent constructs that allowed us to achieve high affinity interaction with the lectin.

CARB 13

Design and discovery of GMI 1070 (Rivipansel), a novel pan-selectin antagonist for the treatment of vaso-occlusive crisis in sickle cell disease

John M. Peterson1, jpeterson@glycomimetics.com, Arun Sarkar1, John T. Patton1, Miti Rahman1, Norman Karasanyi1, Beat Ernst2, John L. Magnani1. (1) Glycomimetics, Inc., Gaithersburg, Maryland, United States (2) Department of Pharmaceutical Sciences, University of Basel, Magden, Switzerland

The selectins are a family of cell adhesion molecules involved in lymphocyte homing and inflammatory processes. The 3 members of the family – E-, P-, and L-selectin – recognize the tetrasaccharide sialyl Lewisx/sialyl Lewisa (sLex/sLea) as a key binding element. P- and L-selectin require the presence of additional sulfated tyrosine residues. The selectins have been implicated in the pathophysiology of vaso-occlusive crisis (VOC) in sickle cell disease (SCD). In order to offer a new treatment modality to SCD patients we desired a pan-selectin antagonist. Key points of contact between sLex and E-selectin have been determined by transfer-NOE and STD NMR experiments, and X-ray crystallography. We have developed a glycomimetic E-selectin antagonist which mimics these key interactions. A substructure designed to bind the sulfate domain of P-, and L-selectin was identified by screening a suramin derived library. Linking the glycomimetic domain with the sulfate domain generated the pan selectin antagonist GMI 1070 (Rivipansel). Rivipansel showed robust activity in a murine model of vaso-occlusive crisis and was advanced into human clinical trials. It was well tolerated in a Phase I study. A Phase II study treating VOC in SCD patients resulted in consistent reductions in the need for narcotic pain relief, the duration of VOC, and the length of hospital stay. The success of Rivipansel in Phase II clinical trials supports the use of glycomimetic compounds as innovative therapeutic strategies to address unmet medical needs.

CARB 14

New NMR tools for unraveling the conformation, dynamics, and recognition properties of glycomimetics

JESUS JIMENEZ-BARBERO1,2, jjbarbero@cicbiogune.es. (1) Structural Biology, CIC bioGUNE, Derio, Bizkaia, Spain (2) Chemical & Physical Biology, CIB-CSIC, Madrid, Madrid, Spain

Molecular recognition by specific targets is at the heart of the life processes. In recent years, it has been shown that the interactions between proteins (lectins, enzymes, antibodies) and carbohydrates mediate a broad range of biological activities, from fertilization, embryogenesis, and tissue maturation, to pathological processes. The elucidation of the mechanisms that govern how sugars and glycomimetics are accommodated in the binding sites of these receptors is currently a topic of interest. Thus, the determination of the structural and conformational factors and the physicochemical features which govern the molecular recognition of these molecules is of paramount importance. This presentation is focused on the application of NMR methods to the study of molecular recognition processes between a variety of glycomimetics with biomolecular receptors. Especial emphasis will be placed in the use of fluorine-containing glycomimetics and sugar derivatives containing lanthanide-binding-tags. Moreover, examples will be shown for dissecting the robustness of the different approaches, with the benefits and drawbacks, together with the experimental requirements, both from the spectrometers and from the sample availability.

CARB 15

Galectin-ligand analogs and mimetics in intracellular vesicle damage and in angiogenesis

Ulf J. Nilsson1, ulf.nilsson@chem.lu.se, Hakon Leffler2, Noorjahan Panjwani3. (1) Department of Chemistry, Lund University, Centre for Analysis and Synthesis, Lund, Sweden (2) Department of Laboratory Medicine, Section MIG, Lund University, Lund, Sweden (3) Tufts University School of Medicine, Boston, Massachusetts, United States

Galectins are glycoconjugate-binding lectins that via cross-linking of galactopyranose containing glycans are believed to influence e.g. glycoprotein trafficking, localization, residence times, which in turn may impact glycoprotein functions in cell proliferation, inflammatory processes, tumor growth, and tumor metastasis.[1] Consequently, this has sparked a growing interest in developing galectin agonist and antagonists as research tools and possibly drug leads.[2] Herein we will discuss our recent efforts towards the discovery and development of high-affinity and selective galectin-antagonizing analogs and mimetics of endogenous galectin ligand fragments with emphasis on aromatic-arginine interactions.[3] In addition, the use of such analogs and mimetics in evidencing the role galectin-3-glycan interactions in VEGFR2 signaling controlling angiogenesis[4] and in intracellular vesicular damage[5] will be presented. [1] aJ. W. Dennis, C. F. Brewer, Mol. Cell. Proteomics 2013, 12, 913-920; bS. Di Lella, V. Sundblad, J. Cerliani, C. M. Guardia, D. A. Estrin, G. R. Vasta, G. A. Rabinovich, Biochemistry 2011, 50, 7842-7857. [2] R. J. Pieters, ChemBioChem 2006, 7, 721-728.

[3] aI. Cumpstey, E. Salomonsson, A. Sundin, H. Leffler, U. J. Nilsson, Chem. Eur. J. 2008, 14, 4233-4245; bI. Cumpstey, A. Sundin, H. Leffler, U. J. Nilsson, Angew. Chem. Int. Ed. 2005, 44, 5110-5112; cB. A. Salameh, I. Cumpstey, A. Sundin, H. Leffler, U. J. Nilsson, Bioorg. Med. Chem. 2010, 18, 5367-5378; dP. Sörme, P. Arnoux, B. Kahl-Knutsson, H. Leffler, J. M. Rini, U. J. Nilsson, J. Am. Chem. Soc. 2005, 127, 1737-1743. [4] W.-S. Chen, H. Leffler, U. J. Nilsson, N. Panjwani, FASEB J. 2013, 27, 828.821. [5] aG. Sharma, J. Stegmayr, M. C. Carlsson, U. J. Nilsson, S. Oredsson, H Leffler, 2014 ASCB-IFCB Meeting Abstract ID: 1751; bJ. Stegmayr, G. Sharma, M. Carlsson, S. Oredsson, U.J. Nilsson, H. Leffler, 2014 ASCB-IFCB Meeting Abstract ID 64.

CARB 16

Druggability of lectins, using the example of a bacterial adhesin

Beat Ernst, beat.ernst@unibas.ch, Priska Frei, Jacqueline Bezencon, Deniz Eris, Said Rabbani, Pascal Zihlmann, Roland Preston, Brigitte Fiege. Department of Pharmaceutical Sciences, University of Basel, Basel, Switzerland

In general, the druggability of lectins (the likelihood of being able to modulate a lectin with a small-molecule drug) is considered to be low. Thus, lectins are still carelessly neglected regarding their therapeutic potential. Using the specific example of the bacterial lectin FimH, we therefore challenged the various pharmacodynamic and pharmacokinetic drawbacks associated with carbohydrate-lectin interactions. FimH is an adhesin involved in urinary tract infections (UTIs), which are primarily caused by uropathogenic Escherichia coli (UPEC). UTIs affect millions of people each year, and account for significant morbidity and high medical costs. These infections are initiated by adhesion of the lectin FimH, located at the tip of bacterial type 1 pili, to oligomannosides of the glycoprotein uroplakin 1A on bladder endothelial cells. Since bacterial resistance to antibiotics is a major problem of recurrent infections, the blocking of bacterial adhesion with soluble carbohydrates or analogs thereof is regarded as a promising therapeutic approach to UTIs. In a detailed analysis, the contributions to binding of various FimH antagonists were analyzed by X-ray, ITC and NMR, leading to a detailed understanding of the binding process. In addition, pharmacokinetic properties were optimized according to the requirements for oral administration. Finally, the most promising candidate was validated in an in vivo disease model, demonstrating its high therapeutic potential for the prevention and/or therapy of UTIs.

CARB 17

Well-defined antibody-drug conjugates (ADCs) through site-specific conjugation

G Boons, gjboons@ccrc.uga.edu. Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia, United States

A new methodology has been developed for the preparation of well-defined antibody-drug conjugates (ADCs) by glycan remodeling using CMP-Neu5Ac modified by an azido function. The enzyme ST6Gal 1 is uniquely suited to install the azido-containing sialic acid into the glycans of IgG antibodies to give homogeneous glycoforms having approximately four azido functions. A cytotoxic drug can be attached to such an antibody by strain promoted azide-alkyne cycloaddition, which is much more attractive than conventional conjugation methods, because it very efficient and selective and does not require a toxic metal catalyst to induce the cycloaddition. The methodology was employed to modify an anti-CD22 antibody with a cytotoxic drug, and the resulting ADC could selectively kill lymphoma cells, which overexpress CD22. The conjugation methodology does not interfere with antibody effector functions. The new method for homogenous ADC production is very attractive because it does not require genetic engineering and therefore is applicable to any type of therapeutic antibody.

CARB 18

Isoform selective inhibition of tumor-associated carbonic anhydrase IX using carbohydrate-based sulfamates for the treatment of several cancers

Brian P. Mahon1, brianpmahon@ufl.edu, Janina Ladwig4, Laurent Bornaghi4, Daniela Vullo2, Sally-Ann Poulsen4, Claudiu T. Supuran2, Robert McKenna3. (1) Biochemistry & Molecular Biology , University of Floirda, Gainesville, Florida, United States (2) Univ Florence Lab Chim Bioorg, Sesto Fiorentino, Italy (3) Univ Florida Colg of Medicine, Gainesville, Florida, United States (4) Griffithy University, Brisbane, Queensland, Australia

In several cancers, metastatic tumors often exhibit areas of hypoxia and a decrease in extracellular pH due a shift in their general metabolism. This metabolic shift alters the tumor milieu inducing tumor cell proliferation, angiogenesis, cell motility, invasiveness, and often resistance to common anti-cancer treatments. As a result, tumors exhibiting this phenotype are directly associated with a poorer prognosis and decreased survival rate in cancer patients. Carbonic anhydrase IX (CA IX), an extracellular zinc metalloenzyme, is overexpressed in hypoxic tumors and has been shown to play key roles in tumor physiology. As such, inhibition of CA IX has shown to be therapeutically advantageous in terms of reducing tumor proliferation and increasing tumor susceptibility to radiation and chemotherapies. In addition CA IX has minimal expression in normal human tissues making it an ideal drug target. However targeting CA IX has been challenging since it shares a close homology to other ubiquitously expressed human isoforms, thus presenting the potential for off-target effects. Here we present a novel class of CA IX selective inhibitors that utilize a carbohydrate or acetylated-carbohydrate fragment conjugated to a sulfamate zinc-binding group, and describe the structure-activity relationships that allow these compounds to preferentially bind to CA IX over the ubiquitously expressed CA II. All compounds selectively inhibited

CA IX in the nanomolar range (>1000-fold better than other CAs), and also exhibit permeability profiles that favor targeting of extracellular CA IX over the cytosolic CAs. The X-ray crystal structures of five of these compounds in complex with an engineered CA IX-mimic and CA II have been determined to 1.7 Å resolution or better, and demonstrate a selective mechanism of binding between CA IX versus CA II. These inhibitors present promising candidates for anti-CA IX drugs and the treatment for several aggressive cancer types.

CARB 19

Synthesis and properties of polyfluorinated carbohydrates

Bruno J. Linclau, bruno.linclau@soton.ac.uk. Chemistry, University of Southampton, Southampton, United Kingdom

Carbohydrates have a typically low affinity with proteins, due to their excessive hydrophilicity. This hampers applications eg as inhibitors, and hence there is a strong interest in the development of carbohydrate mimetics with improved affinity. The ‘polar hydrophobicity’ concept was proposed as an attractive strategy to achieve higher binding affinity for sugars.[1] It involves replacing multiple sugar ring CHOH groups with CF2 groups, and relies on a combination of the higher hydrophobic desolvation energy by perfluoroalkyl groups compared with alkyl groups,[2] and the possibility for the individual polar C–F bonds to engage in attractive interactions with protein residues, with such interactions being negligible in aqueous medium.[3] Importantly, this modification does not alter the shape/size of monosaccharides. Our group has been investigating tetrafluorinated sugars for this purpose,[4] and we recently became interested in sugars having other, less fluorinated hydrophobic motifs. This presentation will explain the background behind this interest, and will give a brief overview of the synthesis and lipophilicity measurements of some of these sugars. A successful example of a tetrafluorinated Galf-UDP inhibitor (to UGM) will be presented.[4e] A summary of our findings on how fluorination influences alcohol hydrogen bond donating properties will also be included. [1] a) H. W. Kim, Paolo Rossi, R. K. Shoemaker and S. G. DiMagno, J. Am. Chem. Soc. 1998, 120, 9082; b) J. C. Biffinger, H. W. Kim and S. G. DiMagno, ChemBioChem 2004, 5, 622. [2] J. Mecinovic, P. W. Snyder, K. A. Mirica, S. Bai, E. T. Mack, R. L. Kwant, D. T. Moustakas, A. Heroux and G. M. Whitesides, J. Am. Chem. Soc. 2011, 133, 14017. [3] a) R. Paulini, K. Müller and F. Diederich, Angew. Chem. Int. Ed. 2005, 44, 1788; b) K. Müller, C. Faeh and F. Diederich, Science 2007, 317, 1881. [4] Key references: a) A. J. Boydell, V. Vinader and B. Linclau, Angew. Chem. Int. Ed. 2004, 43, 5677; b) R. S. Timofte and B. Linclau, Org. Lett. 2008, 10, 3673; c) B. Linclau, S. Golten, M. Light, M. Sebban and H. Oulyadi, Carbohydr. Res., 2011, 346, 1129; d) I. N’Go, S. Golten, A. Arda, J. Canada, J. Jimenez-Barbero, B. Linclau, S. Vincent, Chem. Eur. J. 2014, 20, 106; e) L. Mtashobya, L. Quiquempoix, B. Linclau, J. Fluorine Chem., In Press, Available online 16 September 2014, DOI 10.1016/j.fluchem.2014.08.023.

CARB 20

Computational design of glycomimetic inhibitors - prospects and limitations

Martin Frank1, martin@glycosciences.org, Per-Georg Nyholm2. (1) Biognos AB, Gothenborg, Sweden (2) Comp Chem, Biognos AB, Goteborg, Sweden

The design of molecules that mimic structural features of carbohydrates but have improved drug-like properties such as increased affinity, serum half-life, stability, and bioavailability has gained growing attention over recent years. Here we give an overview on computational methods that can support this endeavor and show some success stories. We also highlight novel glycoinformatic approaches to mine for glycomimetics in commercially available compound libraries which would allow to generate focused libraries for virtual screening.

CARB 21

Sugars and proteins: Building glycoproteins

Benjamin G. Davis, ben.davis@chem.ox.ac.uk. Univ of Oxford Chem Rsrch Lab, Oxford, United Kingdom

Sugars and Post-Translational Modifications are critical biological markers that modulate the properties of Proteins. Our work studies the interplay of proteins, sugars and modifications. This lecture will cover emerging areas in our group in: (a) biomolecule construction with an emphasis on new bond-forming processes compatible with biology & (b) use of chemical probes & modulators of carbohydrate & protein processing events. Synthetic Biology’s development at the start of this century may be compared with Synthetic Organic Chemistry’s expansion at the start of the last; after decades of isolation, identification, analysis and functional confirmation the future logical and free-ranging redesign of biomolecules offers tantalizing opportunities. Glycoproteins represent one of the most challenging targets and opportunities in both Chemistry and Biology. This lecture will consider and compare new methods from our work.

CARB 22

New tools in glycoprotein chemical synthesis

Xuechen Li, xuechenl@hku.hk. Chemistry, The University of Hong Kong, Hong Kong, Hong Kong

Recently, there is an increasing demand to study the protein post-translational modification (PTM) in understanding the increase in complexity from the level of the genome to the proteome. The PTM proteins isolated from the natural sources or obtained from recombinant DNA approaches are characterized by the heterogeneity or the incorrect modification structure, which challenges the following biological studies. In this regard, chemical synthesis of proteins will make important contribution to the production of homogeneous PTM proteins with the high homogeneity and the defined structure. In addition, the chemical synthesis will allow for the generation of new bio-macromolecules with altered structures, including the incorporation of unnatural elements, to create new functions. Our laboratory has a long-standing interest in the study of the protein post-translational glycosylation. At the intial stage, we aim to develop chemical methods to enable us to access the synthetic and homogenous glycoproteins which are homogeneous and of a defined structure. Over the past years, we have established a chemoselective peptide ligation, Serine/Threonine Ligation (STL), enabling the synthesis of biologically active proteins. In addition, we are also engaged in the development of new methods and strategies enabling complex glycan synthesis.

references: 1) Peng, P.; Liu, H.; Gong, J.; Nicholls, J. M.; Li, X.* Chem. Sci. 2014, 5, 3634. 2) Lee, C. L.; Li, X.* Curr. Opin. Chem. Biol. 2014, 22, 108-114 3) Zhang, Y.; Xu, C.; Lam, H. Y.; Lee, C. L; Li, X.* Proc. Natl. Acad. Sci. USA, 2013, 110, 6657. 4) Lam, H. Y.; Zhang, Y.; Liu, H.; Xu, J.; Wong, C. T.; Xu, C.; Li, X.* J. Am. Chem. Soc. 2013, 135, 6272. 5) Wong, C. T. T.; Lam, H. Y.; Song, T.; Chen, G.*; Li, X.* Angew. Chem. Int. Ed. 2013, 52, 10212.

CARB 23

Synthesis of homogeneous glycoproteins and application to biochemical studies and biosimilar comparability analysis

Thomas J. Tolbert, tolbert@ku.edu. Univ of Kansas, Lawrence, Kansas, United States

The heterogeneity of glycoproteins produced in natural systems makes it difficult to conduct biochemical studies and poses problems for the pharmaceutical industry in characterizing glycoprotein therapeutics. The synthesis of homogeneous glycoproteins using a combination of expression in genetically modified yeast and in vitro enzymatic synthesis will be described. Application of homogeneous glycoproteins produced in this manner to physicochemical and biochemical studies of protein glycosylation will be discussed. In addition, the use of homogeneous glycoforms of antibody fragments as a model for biosimilar comparability analysis will be presented.

CARB 24

Site-specific investigation of O-GlcNAc modifications using synthetic proteins

Matthew Pratt, matthew.pratt@usc.edu. University of Southern California, Los Angeles, California, United States

The modification of intracellular proteins by the monosaccharide N-acetyl-glucosamine (O-GlcNAc) is indispensable for life in mammals and contributes to a variety of human diseases. Neurodegenerative diseases are typically associated with the formation of toxic protein aggregates. Notably several of these proteins are O-GlcNAcylated. Previous data have demonstrated that O-GlcNAcylation of the protein tau inhibits its aggregation; however, was yet known about other aggregation-prone proteins. To begin to address this knowledge gap, I will present the use of protein semi-synthesis to first prepare site-specifically O-GlcNAcylated α-synuclein, the major aggregating protein in Parkinson’s disease, and then characterize the consequences of O-GlcNAc on its aggregation and cellular toxicity.

CARB 25

Chemical synthesis as a tool to study protein glycosylation

Zhongping Tan, Xiaoyang Guan, xiaoyang.guan@colorado.edu. Department of Chemistry and Biochemistry, University of Colorado Boulder, Boulder, Colorado, United States

Protein glycosylation is an important post-translational modification. It enhances the functional diversity of proteins and alters their stability, binding affinity, substrate specificity and biological activity. However, because of the complexity of glycoproteins and glycan structures, establishing connections between glycan structures and their effects is difficult. Recently, we demonstrate that chemical synthesis can be employed as a useful tool to determine the effects of protein glycosylation. The more precise understanding of protein glycosylation would aid in the development of enzymes and biotherapeutics with improved performance.

CARB 26

Intracellular traffic of cell surface mimetic quantum dots-anchored glycopeptides

Shin-Ichiro Nishimura1,2, shin@glyco.sci.hokudai.ac.jp, Roger S. Tan3, Kentaro Naruchi2, Maho Amano1, Hiroshi Hinou1. (1) Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan (2) Medicinal Chemistry Pharmaceuticals, Co., Ltd., Sapporo, Japan (3) Grad Sch of Life Sci, Hokkaido Univ, Sapporo, Japan

An efficient method for the construction of cell surface mimetic quantum dots (QDs)-anchored peptides/glycopeptides was established. We present a standardized protocol

for the preparation of multifunctional QDs that allow for efficient cellular uptake and rapid escaping from the endolysosomal system, subsequent cytoplasmic molecular delivery to the target cellular compartment. Chemoselective ligation of the ketone-functionalized hexahistidine derivative allowed for both endocytic entry and rapid endolysosomal escape of the aminooxy/phosphorylcholine self-assembled monolayer-coated QDs (AO/PCSAM-QDs)1 to the cytosol in various cell lines such as human normal and cancer cells. Combined use of hexahistidylated AO/PCSAM-QDs with serglycine-like glycopeptides, namely synthetic proteoglycan initiators, elicited the entry, controlled intracellular trafficking, Golgi localization, and also excretion of these nanoparticles, suggesting that the present approach will provide an ideal platform for the design of high performance nanoparticle-based drug delivery vehicles systems.

CARB 27

Protein glycosylation in the baculovirus-insect cell system

Donald L. Jarvis1,2, dljarvis@uwyo.edu. (1) Molecular Biology, University of Wyoming, Laramie, Wyoming, United States (2) GlycoBac, LLC, Laramie, Wyoming, United States

The baculovirus-insect cell system is widely used to produce recombinant glycoproteins for diverse biomedical research projects and direct clinical applications. However, there are some key differences between insect and higher eukaryotic cellular protein glycosylation pathways, which can effectively preclude the use of recombinant glycoproteins produced in the baculovirus-insect cell system for some applications. One difference is that insect cell lines are generally unable to elongate trimmed N- and O-glycans to produce complex, terminally sialylated structures. Another is that some insect cell lines can produce highly immunogenic, core alpha-fucosylated N-glycans. cannot synthesize products with structurally authentic carbohydrate side chains. Our research focuses on efforts to elucidate and engineer protein glycosylation pathways in the baculovirus-insect cell system. This presentation will provide an overview of our recent glycoengineering efforts, which have yielded new baculovirus-insect cell systems with the ability to produce humanized, non-immunogenic recombinant glycoproteins.

CARB 28

Manipulating protein stability by glycosylation

Yaakov (Koby) Levy, koby.levy@weizmann.ac.il. Weizmann Institute of Science, Rehovot, Israel

The biophysical characteristics of proteins can be affected by either in vivo or engineered modifications. In my talk, I will focus how computational chemistry can assist in understanding the effect of glycosylation and PEGylation on the thermodynamic stability of proteins. The stability may dictate many other features of the protein, such as its structural integrity, its fluctuations and thus its function. However, predicting protein stability is a computationally challenging task that is evident from the

fact that protein stability is limited and is often of few kcal/mol only, which is due to a balance between several opposing contributions (entropic and enthalpic components of either the folded or unfolded states). Common means to change protein stability includes point mutations or the molecular properties of the solvent. In my talk, I will discuss several examples in which conjugating the protein to a polymer (for example, carbohydrate in glycosylation or PEG in PEGylation) may affect stability. The biophysical consequences of the tethering can vary depending on the molecular nature of the conjugate as well as the conjugation site. Furthermore, the molecular origin of the effect of conjugation on stability is sometimes non-trivial and can be either enthalpic or entropic. I will discuss how glycans can be used to modulate protein characteristics depending on the molecular properties of the glycans itself and the modifications sites on the proteins. Our studied proteins illustrate not only how protein stability can be modulated but also how it is evolutionarily controlled for biological function.

CARB 29

Impact of glycosylation upon protein conformational tendencies

William G. Noid, wnoid@chem.psu.edu. Pennsylvania State Univ, University Park, Pennsylvania, United States

N-linked carbohydrates can dramatically impact the structure and thermodynamic stability of glycoproteins. In this talk we present computational investigations of this effect for short peptides. In particular, we employ extensive replica exchange molecular dynamics simulations of atomically detailed models with explicit solvent to elucidate the precise interactions that trigger a glycosylation-induced conformational switch that has been experimentally observed for a model octapeptide with the sequence Ile-Thr-Pro-Asn-Gly-Thr-Trp-Ala. The simulations suggest that hydrogen bonding and aromatic-glycan stacking interactions both contribute to stabilize the switch from an extended, disordered ensemble to a much more compact ensemble with beta hairpins. They also suggest that steric crowding by the N-linked disaccharide is inadequate to trigger this switch and that the glycan appears to have relatively little impact upon the local water structure. Moreover, we demonstrate that, for this peptide, the simulated conformational switch is remarkably sensitive to steric clashes with the sidechain adjacent to the glycosylation site, but less sensitive to aromatic-glycan stacking interactions. Finally, we demonstrate that these simulated observations are remarkably consistent with the known structures of full length glycoproteins in the Structural Assessment of Glycosylation Sites database.

CARB 30

Computational study of glycosylphosphatidyl-Inositol (GPI) anchor fragments embedded in phospholipid membranes

Marko Wehle1, Reinhard Lipowsky1, Peter H. Seeberger2, Gerald Brezesinski3, Cristina Stefaniu3, Daniel Varon-Silva2, Mark Santer1, mark.santer@mpikg.mpg.de. (1) Theory

and Bio Systems, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam-Golm, Germany (2) Dept of Biomolecular Systems, Max Planck Institute of Colloids Interfaces, Berlin, Germany (3) Department of Interfaces, Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Brandenburg, Germany

We present a numerical study of several types of fragments of the Glycosylphosphatidylinositol(GPI-) anchor embedded in lipid membranes and as pure Langmuir monolayers. The glucosamine α1-6myoIno-1-PO4 unit of the glycan core of the GPI has been parametrized in the GLYCAM framework and we show that it can be connected smoothly to the new models for phospholipids provided as part of the AMBER force field library (LIPID14). This hybrid model for a glycolipid will be explored in several respects. One emphasis is on how the carbohydrate backbone of the GPI is embedded within the hydrophilic head group part of a membrane under different conditions, such as different partial charge (GLYCAM vs. AMBER)- and water models (e.g. TIP3P and TIP5P). We also present comparisons to experimental results, such as the diffusivity of GPI fragments with varying core length, or the strong network of intermolecular hydrogen bridges found in GPI monolayers. This network leads to pronounced sub-gel phases which can be only suspended at high concentrations of urea [1]. We will discuss the general implications of our results for GPI-anchored proteins. 1. C. Stefaniu, I. Vilotijevic, M. Santer, G. Brezesinski, P. H. Seeberger and Varόn-Silva, D. Versatility of a GPI-fragment in forming highly ordered Polymorphs. 2014. Langmuir 30(18):5185—5192.

CARB 31

Polysaccharides from lactic bacteria: interactions and complexation with proteins and nanoparticles

Louise Deschenes1, louise.deschenes@agr.gc.ca, Nancy Guertin2, François Saint-Germain3, tony savard4, Denise Chabot3. (1) Agriculture Agri-Food Canada, Food Research and Development Centre, Saint-Hyacinthe, Quebec, Canada (2) Agriculture and Agri-FOod Canada, Saint-Hyacinthe, Quebec, Canada (3) agriculture and Agri-Food Canada, Saint-Hyacinthe, Quebec, Canada (4) Agriculature and Agri-Food Canada, Saint-Hyacinthe, Quebec, Canada

Polysaccharides are important extracellular components produced by lactic bacteria. Aside from their production by planktonic cells, these polycarbohydrates are involved in several mechanisms such as cell protection and biofilm formation, hence essential to bacteria activities and survival. In a previous study, it was shown that (ZnO-NPs) partially dissolve in MRS growth media and that the survival of lactobacillus was ZnO and Zn2+-dependent. It was also observed that ZnO-NPs are associated to aggregates which literally surround and smoother bacteria. These aggregates are believed to be polysaccharide-mediated. The present work was undertaken to develop a better understanding of the role of polysaccharides and aggregates formation in presence of

ZnO and Zn2+. Two EPSac were selected for the study: a negatively charged produced by Lactobacillus casei LB31 and a neutral one produced by CNRZ371. Tensiometric measurements indicated that these EPSac are not surface-active. They both have negative zeta potential in 10 mM bis-tris-propane pH7 and in acetate buffer, pH5. Interactions between lactobacillus EPSac and proteins in presence of divalent and monovalent salts were investigated by dynamic light scattering (DLS). The effect of the charged EPSac on ZnO nanoparticles suspensions was also monitored by DLS. At a concentration of 2 mg/ml, both EPSacs presented a polydispersed distribution in aqueous solutions. A larger proportion of large aggregates were observed in presence of divalent ions in mixtures of the negatively charged EPSac and BSA.

CARB 32

Synthesis and antibacterial activity of antibiotic-functionalized graphite nanofibers

Robert M. Giuliano, robert.giuliano@villanova.edu, Madeline Rotella, Alicia Briegel, John Hull, Anthony F. Lagalante. Villanova Univ, Villanova, Pennsylvania, United States

Surface functionalization of nanomaterials is an area of current investigation that supports the development of new biomaterials for applications in biology and medicine. Herein, we describe the synthesis, characterization, and antibacterial properties of the first examples of graphite nanofibers (GCNFs) that have been covalently functionalized with aminoglycoside and quinolone antibiotics. Ruthenium tetroxide oxidation of herringbone GCNFs gave higher amounts of surface carboxyl groups than previous methods. These carboxyl groups served as sites of attachment for antibiotics by acyl substitution. Bioassay of these novel, functionalized GCNFs using serial dilution and optical density methods demonstrated that antibiotic-labeled GCNFs possess antibacterial activity against Pseudomonas aeruginosa. The acitivity we observe for aminoglycoside-functionalized GCNFs suggests a membranolytic mechanism of action.

CARB 33

Encapsulation and release of an active enzyme utilizing cobalt crosslinked chitosan nanoparticles

Jana B. Lampe2, janalampe@me.com, Gerardo Castillo2, Clifford S. Morrison2, Duong Nguyen3, Ronaldo J. Cavazos2, Robby A. Petros1. (1) CB 305070, UNT Dept of Chemistry, Denton, Texas, United States (2) Chemistry, University of North Texas, Denton, Texas, United States

Targeted delivery of proteins represents an attractive goal for treating a variety of diseases caused by enzymatic disorders, such as Gaucher’s disease. Here we report the encapsulation and release of a prototypical enzyme, lysozyme, using chitosan nanoparticles. The bioconjugation strategy utilized cobalt coordination chemistry to crosslink amine residues in chitosan and the protein. Particle sizes 10-500 nm could be

trivially synthesized by adjusting the relative concentrations of the components used in particle synthesis. The cobalt bioconjugation chemistry was impervious to water, and required no specialized reagents or procedures. The process relies on self assembly of particles in the presence of exchange labile Co2+ that could be locked into configuration by oxidation to exchange inert Co3+. In order for such an approach to be successful, the protein must be released in its native, enzymatically active state. Encapsulation of lysozyme in chitosan nanoparticles led to almost complete amelioration of its enzymatic activity that could be reversed upon exposure of the nanoparticles to reductant (glutathione) at physiologically relevant concentrations. These results make this novel bioconjugation strategy highly attractive for the targeted delivery of other therapeutic enzymes and proteins. Details of particle synthesis, characterization, and enzyme activity will be presented.

CARB 34

Reduction of flammability of cotton fabrics treated with phosphoryl piperazine derivatives

Thach-Mien Nguyen 1, phip777@yahoo.com, Sechin Chang1, Brian Condon1, Ryan Slopek2, Elena Graves1. (1) U.S. Department of Agriculture, Metairie, Louisiana, United States (2) USDA-ARS-SRRC, New Orleans, Louisiana, United States

The use of flame retardants (FRs) for textiles has undergone a significant evolution due to environmental issues related to the use of certain types of high-performing FRs. As a result in the last 30 years, many of the high-performing formaldehyde- or halogen-based FRs for fabrics have been banned from commercial use, thus favoring the use of phosphorus-containing products. In this study, a phosphoryl-piperazine derivative O,O,O',O'-Tetramethyl piperazine-1,4-diyldiphosphonothioate (TMPT) was modified from Tetraethyl piperazine-1,4-diyldiphosphonate (TEPP) and the effectiveness as flame retardants of these two compounds for cotton fabric was obtained and compared. TMPT differs from TEPP by a substitution of sulfur for oxygen and a reduction of one carbon in phosphorus moiety. The former was prepared in a simple and effective one-step reaction giving an excellent yield (93 %) of product. Cotton fleece, twill and print cloth were then treated with both compounds to provide different add-on values. Thermal degradation and flammability of the control and treated fabrics were investigated by using thermogravimetric analysis (TGA), and the flammability tests such as vertical, 45 degree angle, limiting oxygen index (LOI). More thorough and focused studies were done by identifying the functional groups which appeared on the surface of the control and treated twill fabrics using attenuated total reflection-infrared spectroscopy (ATR-IR), investigating the released gas products using thermogravimetric analysis-fourier transform infrared spectroscopy (TGA-FTIR) and examining the microstructures of unburned and burned areas using scanning electron microscopy (SEM). The results showed better flame retardancy and thermal behavior for TEPP when compared to TMPT and they revealed some significant differences in the degradation of the treated fabrics during thermal decomposition process.

CARB 35

Synthesis of hyaluronic acid-based phototherapeutics

Renato A. Guerrieri1, reguerrieri@davidson.edu, Erin Xu1, erxu@davidson.edu, Ryan D. Dolewski2, Rachel G. Barkley1, Joshua V. Ruppel2, Nicole L. Snyder1. (1) Chemistry, Davidson College, Davidson, North Carolina, United States (2) Division of Natural Sciences & Engineering, University of South Carolina Upstate, Spartanburg, South Carolina, United States

Recent studies have examined the use of hyaluronic acid (HA) drug conjugates for the diagnosis and treatment of different types of cancers including lung, bladder, pancreatic, ovarian, breast and colon cancers, as well as various melanomas, carcinomas and sarcomas. Here we report on our work on the synthesis of a new class of HA-based drug conjugates that have the potential to be used in photodynamic therapy.

CARB 36

Synthesis of meso-substituted carbohydrate porphyrin and carbohydrate bacteriochlorin conjugates

George T. Mukosera1, gemukosera@davidson.edu, Ryan D. Dolewski2, Joshua V. Ruppel2, Nicole L. Snyder1. (1) Chemistry, Davidson College, Davidson, North Carolina, United States (2) Division of Natural Sciences & Engineering, University of South Carolina Upstate, Spartanburg, South Carolina, United States

We are interested in the synthesis of carbohydrate porphyrin and carbohydrate bacteriochlorin analogs which can be used as water soluble phototherapeutics. Our initial goal was to conjugate an azido sugar to a porphyrin or bacteriochlorin bearing an alkyne handle using a 1,3-dipolar cycloaddition reaction. This reaction has been used in the past to successfully conjugate carbohydrates to porphyrins and bacteriochlorins via linkers or through the aryl rings on the porphyrin or bacteriochlorin macrocycle. Our efforts to link azido sugars directly to meso-substituted alkynyl porphyrin and bacteriochlorin conjugates resulted in low yields and the production of an unwanted oxidation byproduct. In this report, we describe our efforts to rationalize these outcomes using a series of chemical and computational experiments. Our initial findings reveal that by carefully controlling the reaction conditions we may be able to optimize desired reaction products while minimizing unwanted side products.

CARB 37

Progress on the total synthesis of Aspergillus fumigatus galactosylaminoglycans for diagnostic and therapeutic applications

Erich J. Baker, erbaker@davidson.edu, Nicole L. Snyder. Chemistry, Davidson College, Davidson, North Carolina, United States

Aspergillus fumigatus is an opportunistic fungal pathogen. Immunocompromised individuals infected with A. fumigatus can develop a severe respiratory infection known as aspergillosis, which has a mortality rate of 65% despite current antifungal interventions. Due to the high incidence and mortality of aspergillosis, there is a pressing clinical need for effective diagnostics and therapeutics for A. fumigatus. A. fumigatus secretes a unique exopolysaccharide composed of N-acetylgalactosamine residues connected via α-1,4 glycosidic bonds. This exopolysaccharide, known as galactosaminogalactan (GAG), has been shown to mediate biofilm-based adherence in the respiratory system. Mutant strains of A. fumigatus lacking the gene to produce GAG are not pathogenic. GAG is unique to fungi of the genus Aspergillus and is a key mediator of pathogenicity rendering it an excellent target for Aspergillus specific diagnostics and therapeutics. Here we report our progress on the chemical synthesis of short GAG analogs for diagnostic and therapeutic applications.

CARB 38

Progress on the synthesis of N-acetyllactosamine (LacNAc) probes for studying binding differences carbohydrate recognition domains of galectins-1, -3 and -9

Coraline Tao, zhtao@davidson.edu, Nicole L. Snyder. Chemistry, Davidson College, Davidson, North Carolina, United States

Galectins are a family of carbohydrate binding proteins that are characterized by an evolutionarily conserved carbohydrate recognition domain (CRD) and an affinity for carbohydrate constructs containing β-galactosides such as poly-N-acetyllactosamine or LacNAc. Members of the galectin family decode and interpret the glycome. Preliminary research suggests that the individual biological roles of different members of the galectin family are dependent on their β-galactoside binding affinity. However, significant work remains to determine the specific factors that govern the differences in carbohydrate recognition between different members of the galectin family. The primary goal of our research is to rationally design a series of LacNAc probes that can be used to study how differences such as length and sulfation play a role in carbohydrate recognition in the CRDs of galectins-1, -3 and -9. Galectins-1, -3, and -9 were chosen because they are representative of the three galectin classes (prototype (galectin-1), chimeric (galectin-3) and tandem-repeat (galectin-9)), and they are well characterized. Here we present our initial work in this area, including the design, synthesis, and evaluation of several LacNAc probes.

CARB 39

Carbohydrate-based small molecules with immunostimulatory properties

Cecilia E. Marzabadi1, cecilia.marzabadi@shu.edu, Vikram Basava1, Constantine Bitsaktsis2, Emi Hanawa1. (1) Chemistry Dept, Seton Hall University, South Orange, New Jersey, United States (2) Department of Biological Sciences, Seton Hall University, South Orange, New Jersey, United States

Immunostimulatory molecules are useful in a range of applications from vaccine adjuvants to chemotherapeutic agents. Recently we have synthesized some simple saccharides that possess the property of activation of innate immunity in cells. The synthesis and properties of some of these molecules will be described. The mechanism by which we believe they activate innate immunity will also be presented.

CARB 40

Synthesis of wooden based resource derived furanic diol and polymerization of PU via various isocyanate

Baek-Jin Kim2, bjkim@kitech.re.kr, SunHee Kim2, Jin Ku Cho1. (1) Green Proces R&D Department, Korea Institute of Industrial Technology, Cheonan Chungnam, Korea (the Republic of) (2) Green Materials and Process R&BD Group, KITECH, Cheon Ahn, Korea (the Republic of)

Polyurethane has been widely used in various industries such as automotive, coating materials and footwear. Many types of petroleum based polymer resins are adopted coating materials for automotive interior part, door trim, instrument panel etc. Some global companies interested in the bio-based coating materials derived from renewable resources. BASF have been developed new polyester-/polyether type polyol for polyurethane coatings materials to reduce the Green House Gas (GHG) and replace petroleum with bio-based resources. We have been studied environmental furan based polyurethane using 2,5-furandicarboxylic acid (FDCA) derived from wooden based Hydroxylmethylfurfural (HMF). In our study, we firstly synthesized polyol using FDCA and dodecanediol and then we additionally react with acrylic diol via allyl cyclic type isocyante. Finally we investigate that the mole ratio of two kinds of diols influence on molecular weight and their film properties such as adhesion strength, abrasion resistance for automotive interior part.

CARB 41

Selective hydrogenation of biomass-derived sugars using supported Ru nanoparticles based catalysts

Jin-Soo Hwang, jshwang@krict.re.kr, Aasif A. Dabbawala. Korea Research Institute of Chemical Technology, Daejeon, South Korea, Daejeon, Daejeon, Korea (the Republic of)

Hydrogenation of Biomass-derived sugars produces various value added chemicals that are potential sustainable substitutes for petroleum-based building blocks used in the production of renewable chemicals. In this view, the present investigation is emphasized on hydrogenation of biomass derived sugars to sugar alcohols. Nanoporous hypercross-linked polystyrene/divinylbenzene copolymer supported Ru catalysts are prepared via chemical reduction wherein ruthenium nanoparticles are dispersed on

organic cross-link polystyrene polymers bearing non-coordinating tertiary amine moieties (Ru/AFPS). The polymer supported Ru nanoparticles based catalysts are characterized using different techniques such as X-ray powder diffraction (XRD), transmission electron microscopy (TEM), energy dispersive X-ray (EDX) analysis, scanning electron microscope (SEM), X-ray photoelectron spectrum (XPS) analysis, thermogravimetric analysis (TGA) and CO chemisorptions. The catalytic activity of Ru-based catalysts, (Ru/AFPS) was assessed in the hydrogenation of glucose to sorbitol under aqueous phase condition. The Ru nanoparticles are stabilized and dispersed on amine-functionalized PS matrix (Ru/AFPS) enhanced the formation of desired product sorbitol. The various reaction parameters were optimized in order to achieve maximum conversion, yield and selectivity to desire product. The catalyst Ru/AFPS is also recycled without significant loss of activity and selectivity.

Hydrogenation of Bio-mass derived Glucose to Sorbitol using Ru/AFPS catalyst

CARB 42

Hydrothermal treatment of eucalyptus using acidic ionic liquid as catalysis toward a biorefinery concept

Ji-Kun Xu1, jikun_xu@163.com, Runcang Sun2. (1) Beijing Forestry University, Beijing, China (2) South China Univ of Tech, Guangzhou Guangdon, China

The application of the acidic ionic liquid (IL) 1-butyl-3-methylimidazolium hydrogensulfate ([bmim]HSO4) as catalysis in the hydrothermal treatment (HTT) and fractionation of lignocellulosic biomass has been investigated. The influences [bmim]HSO4-catalyzed HTT of eucalyptus by determination of sugar and inhibitor formation in the liquid fraction, and chemical and morphological changes of the pretreated solid material coupled with an evaluation of enzymatic hydrolysis. The novel process was carried out in a microwave reactor system at a maximal temperature (140–

200 °C). The liquid fractions were analyzed by HPLC, GPC, and HPAEC. Themorphology and composition of the solid residues were characterized using an array of techniques, such as SEM, XRD, XPS, BET surface area, and CP/MAS 13C NMR. Using a variety of tools, the scientific examination is fundamental toward molecular-level understanding of how the IL-catalyzed HTT process affects biomass structure, cell wall components and cellulose properties that impact on its digestibility. These results provided new insights into the factors limiting enzymatic digestibility and mechanism of biomass deconstruction during this IL-catalyzed hydrothermal process.

CARB 43

Carbohydrate polymer-coating chemistry for cellulose based bioassays

Rong Cao, caorong_chem@hotmail.com, Liyun Guan, Miaosi Li, Wei Shen. Monash University, Melbourne, Victoria, Australia

Herein we studied the exploitation of carbohydrate polymers to prepare a poly interface on cellulose paper for biomolecule conjugation. As a potential platform for point-of-care clinical analyses and environmental diagnostics, cellulose paper-based bioanalytical devices have sparked growing attention. Until now, different strategies to immobilize biomolecules onto paper support have been studied. In this work, we demonstrate the functionalization of the cellulose paper surface with two typical carbohydrate polymers (i.e., chitosan and sodium alginate) respectively, which enables cellulose paper to bear commonly used functional groups for bioassays. Then in order to improve the understanding about the morphology and immobilized biomolecule performance on polysaccharide modified paper biosensing platform, protein immobilization studies were performed. The interfaces between biomolecules and modified cellulose fibres via different methods were characterized by fluorescence microscope and scanning electron microscopy. The developed polysaccharide-coating chemistry is expected to open new approaches for using functionalized cellulose in bioassays like immunodiagnostic applications.

Schematic representation of the carbohydrate polymer modified paper-based biosensing platform

CARB 44

Synthesis of fluorogenic probes for selective biomass degradation by fungi

Qian Zhang1, zhangq1024@gmail.com, Xinrui Peng3, Michelle Grilley3, Jon Takemoto3, Cheng-Wei T. Chang2. (1) Chemistry and Biochemistry, Utah State University, Logan, Utah, United States (2) Utah State Univ, Logan, Utah, United States (3) Biology, Utah State University, Logan, Utah, United States

A library of fifteen commercially purchased and synthetic fluorogenic probes were employed for the investigation of biomass degradation using extract of white-rot fungi. These probes were selected or designed to mimic the dominant linkages in celluloses, hemicelluloses and lignin, three most abundant polymers found in biomass. The results show that white rot fungi display high preference for cleaving mannose-, xylose- and glucose-based probes, which mimic hemicelluloses. Little cleavages were noted for cellobiose-based probes, which mimic celluloses. No cleavage was observed for probes that mimic the linkages in lignin. Overall, these discoveries prove that it is possible to employ fungi for selective degradation or releasing hemicelluloses from biomass.

CARB 45

Synthesis and stability study of DNA duplexes with 1’-carboxamide residues

Wenzhao Dong, wdong@crimson.ua.edu, Stephen A. Woski. Dept of Chemistry Box 870336, Univ of Alabama, Tuscaloosa, Alabama, United States

Deoxyribonucleoside 1’-carboxamidesare being developed as a general method for the introduction of novel functional and reporter groups into DNA oligonucleotides. The design strategy maintains the structure of phosphate groups and 2’-deoxyribofuranosyl ring system, and incorporates nucleobase replacements via an amide linkage at a 1’-carboxylic acid moiety. As a proof of concept, four monomers containing 4-methoxyphenyl,4-nitrophenyl, 4-cyanophenyl and 4-chlorophenyl groups were prepared. All of these were converted to the corresponding phosphoramidite reagents for solid phase oligodeoxynucleotide synthesis. The hybridization of the modified DNAs with complementary strands was studied where the non-natural residues were paired with a natural nucleobase (a thymine) or with a stable abasic residue. In addition, hybridization where no complementary residue is used (a formal “bulge”) was also examined. The resultsof thermal denaturation experiments indicate variant substitutions can be incorporated into oligonucleotide duplexes. Notably, the“bulged” complexes were most stable, suggesting the intercalation of the non-natural residues into the DNA helix. This suggests that the carboxamide motif may be a general method for the insertion of non-natural residues into DNA for applications such as spectroscopic probes.

CARB 46

Semisynthetic approach to cancer vaccines utilizing mimetics of natural and unnatural Tn antigens

Sharmeen Nishat1, nishatsharmeen@yahoo.com, Ahmad A. Shaik1, Peter R. Andreana2. (1) Chemistry, University of Toledo, Toledo, Ohio, United States (2) MS 602, The University of Toledo, Toledo, Ohio, United States

A highly selective glycosylation strategy has been developed to access all stereochemical combinations of tumor associated Thomsen-nouveau (Tn) antigen D-GalNAc-O-Ser/Thr. Phthalimido protected D- or L- amino acid acceptors were utilized in glycosylation yielding α- or β- anomers in excellent yields ranging from 64-96% and inclusive of 100% anomeric selectivity when the appropriate sugar C-2 substitution was installed. Reagent controlled pathway selectivity using Pd-C debenzylation followed by treatment with hydrazine hydrate or hydrazine hydrate alone led to Tn-based carboxylates or the corresponding hydrazides respectively. Hydrazides were selectively condensed with aldehyde functionality of oxidized PS A1 (a zwitterionic polysaccharide isolated from Bacteroides fragilis, known to elicit T-cell dependent immune response) forming hydrazone linkage that allowed synthesis of linker free vaccine constructs to eliminate any unwanted immune response that could be elicited by linker. The long term goal with eight different stereomers of Tn based cancer vaccines is to study the immune responses generated by the synthesized vaccines and therefore compare the effect of stereochemistry on the immunogenicity of the vaccines.

CARB 47

Chemical synthesis and O-glycosidic linkage conformation in a 13C-labeled βMan(1→4)βXyl(1→4)βMan(1→4)βXylOCH3 tetrasaccharide: Effects of linkage structure and context

Wenhui Zhang, zhangwh80@gmail.com, Anthony S. Serianni. Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, United States

Recent studies by Duman and coworkers have revealed the presence of a novel glycolipid in the Alaskan beetle, Upis ceramboides, that possesses potent thermal hysteresis properties (Walters et al., Proc. Natl. Acad. Sci. 2009, 106, 20210-20215). This antifreeze glycolipid (AFGL) is comprised of alternating βXylp and βManp residues connected by (1→4) O-glycosidic linkages 1, although branching and covalent modification of this core structure may also occur. To study the preferred conformations about the φ and ψ torsion angles in the two different O-glycosidic linkages in 1, four singly 13C-labeled disaccharides (21’, 22’, 31’ and 32’) and a triply 13C-labeled tetrasaccharide 4 were prepared chemically. Multiple JCH and JCC values across the “isolated” linkages in 2 and 3, and across the “in-context” linkages in 4 were measured by 1H and 13C NMR spectroscopy, respectively. The trans-O-glycoside J-couplings

(Bose-Basu et al., J. Am. Chem. Soc. 1998, 120, 11158-11173) were fit by a new software program, MA’AT©, to deconvolute the redundant J-couplings into conformatinal models for the φ and ψ torsion angles comprising each glycosidic linkage. The results show that (a) context plays a minimal role in determining linkage conformation in 4, and (b) the two types of β-(1→4) linkages in 4 are conformationally distinct.

CARB 48

Quantitative evaluation of D-galactose–lectin binding properties via development of diversely presented carbohydrate surfaces

Bo Meng, bmeng@ion.chem.utk.edu, Kathrin Tscherch, Michael D. Best, David C. Baker. Department of Chemistry, University of Tennessee, Knoxville, Tennessee, United States

Carbohydrates, which make up a major part of the glycocalix of cell surfaces, play a significant role in governing biological activities such as cell–cell communication and adhesion, host–pathogen interactions and immune defense. Discerning the precise mechanisms by which cell-surface sugars interact with external entities is a scientific challenge. The development of synthetic glycoconjugate surfaces to imitate the natural carbohydrate landscape on the cell surface could shed light on understanding the structure–affinity relationships among carbohydrates in recognition by protein receptors. Herein, we report the construction of microarrays with a collection of diversely presented α-D-galactoside monomers. An α-D-galactose monosaccharide, α-linked 1→2-, 1→3-, 1→4- and 1→6-D-galactose disaccharides, and an α-linked D-galactose trisaccharide, all constructed with propargyl aglycons, were synthesized in a modular fashion. They were immobilized to an azide-coated glass slide with tethering by click chemistry to mimic the carbohydrate presentations featured on cell surfaces. Several fluorescent-labeled lectins were interrogated to determine their recognition with our varied synthetic “cell surface” landscapes and quantitatively analyze their binding affinities and dissociation constants. This study is designed to add new dimensions to our understanding of the effects of sugar density, avidity, and steric configuration of carbohydrate ligands in carbohydrate–lectin binding.

CARB 49

Lipase-mediated modification of peracylated macrolactonic sophorolipids

Aliya Sembayeva, asembayeva@nu.edu.kz, Jason A. Carr. Department of Chemistry, Nazarbayev University, Astana, Kazakhstan

Chemoenzymatic modification of well-defined macrolactonic sophorolipid (SLML) analogues via a series of successive regioselective deesterification/transesterification reactions is investigated. This study represents, to the best of our knowledge, the first careful evaluation of lipase-catalyzed reactions of macrolactonic sophorolipid derivatives. Of the lipases screened, Candida antartica (Novozym 435, carrier fixed lipase fraction B) successfully deacylated the C-6′ of peracylated SLML in anhydrous THF (Scheme 1) and subsequent transesterification with acylating agents (esters of fatty acids) yielded well-defined analogues of varying amphilicity (Scheme 2). This study also shows that the macrolactonic motif is essential for enzymatic recognition of the sophorose rings of these complex glycolipids. In the absence of the lactonic motif, the peracylated sophorose rings are not deacylated, rather the carboxyl end of the non-lactonic forms was preferentially transesterified.

CARB 50

Optimization of autohydrolysis of bamboo for the production of low-DP xylo-oligosaccharides using response surface methodology

Xiao Xiao1, bigxiao321@gmail.com, Jing Bian2. (1) Beijing Key Laboratory of Lignocellulosic Chemistry, Beijing, China (2) Beijing Forestry University, Beijing, China

Bamboo powder was treated with hot water in non-isothermal conditions for various reaction times to generate xylo-oligosaccharides in an autoclave with mechanical stirring. The influence of reaction conditions on the yield, compositions and characteristics of low-DP xylo-oligosaccharides (DP 2-6) were evaluated by using response surface methodology. The optimal reaction condition for the production of low-DP xylo-oligosaccharides was obtained, which were reaction temperature 179 ºC,

reaction time 32 min. Meanwhile a relatively low level of xylose and other products (arabinose, galactose, HMF, furfural, acetic acid and formic acid) was observed, which was conductive to the further purification and utilization. The result demonstrated that the yield of low-DP xylo-oligosaccharides obtained by autohydrolysis depends on the reaction conditions, especially reaction temperature and time.

CARB 51

Facile synthesis of nested fragments of high-mannose N-glycans with lightly protected glycosyl acceptors

Qingfeng Pan1, qpan@omicronbio.com, Shikai Zhao1, Wenhui Zhang1,2, Zongren Zhang1, Anthony S. Serianni2. (1) Omicron Biochemicals, Inc., South Bend, Indiana, United States (2) Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, United States

Cell-surface oligosaccharides appended to proteins and lipids are key binding epitopes in many critical biological processes, including bacterial infection, cell development and the immune response. Understanding these processes at the molecular level requires access to a wide range of oligosaccharides of known structure to support investigations of enzyme substrate binding and specificity, to screen for carbohydrate binding proteins, and to develop assays for enzymic activity. These oligosaccharides are currently unavailable in the commercial sector, thus impeding progress in this field. To address this deficiency, a range of nested fragments of the parent high-mannose oligosaccharide (14-mer), which is transferred en-bloc during protein translation, was synthesized. More than thirty high-mannose oligosaccharides ranging in size from tri- to hexa-saccharides were prepared. Lightly-protected mannosyl acceptors were utilized to facilitate these syntheses. Each oligosaccharide was prepared in multiple milligram or greater quantities and in high purity, as determined by NMR, high-resolution mass spectrometry, and high-pressure liquid chromatography. Samples will be used by the NCI to assemble glycan arrays to screen for carbohydrate binding proteins, and for other biochemical or biomedical applications. This work was supported by NIH/NCI SBIR Contract HHSN261201300038C.

CARB 52

Synthesis of 2-amino sugar building blocks and application for glycodiversification studies

Christian M. Rojas, Ashley Brown, arb2194@barnard.edu, Ginika Ezeude, gie2000@barnard.edu, Margeaux Miller, Amber Scharnow, Alexandria Oviatt. Chemistry Department, Barnard College, New York, New York, United States

Rhodium- and copper-catalyzed amidoglycosylation reactions of glycal 3-carbamates provide synthetic access to stereochemically varied 2-amino sugar derivatives that

contain a cis-fused 2N,3O-oxazolidinone moiety. Functionality for further chemoselective elaboration, including azido and alkynyl groups, can also be incorporporated within the 2-amino sugar products. Additionally, the corresponding free reducing sugars are readily accessible from the initial amidoglycosylation products. Using this synthetic methodology, we have prepared a range of 2-amino sugars for glycodiversification studies and are exploring their utility in coupling reactions with aglycons.

CARB 53

Novel catalytic approach for the regioselective oxidation of carbohydrates under mild conditions

Wataru Muramatsu, muramatu@nagasaki-u.ac.jp. Graduate School of Biomed Sci, Nagasaki University, Nagasaki, Japan

Carbohydrates are important molecules that attracted much attention in various research fields because of their particular and various bioactivities. In particular, thiosaccharides have been demonstrated to be involved in biological phenomena such as adhesion, proliferation, and apoptosis. The goal of this study was to develop a synthetic route based on the use of an organotin catalysts to generate ketosaccharides, which are precursors of the biologically important pseudo saccharides, with high regioselectivity. Using Oc2SnCl2 as the organotin catalysts in the presence of trimethylphenylammonium tribromide, I achieved the regioselective oxidation at the axial-OH group of 1,2-diol moieties of galactopyranosides under mild conditions, which could also be used for the regioselective oxidation of several unprotected carbohydrates.

CARB 54

Using potatoes as a carbohydrate based additive in road salt

Richard Byrnes1, richard.byrnes@maine.edu, Donald Szlosek2, Greg Smith1, Joe Walters1, Rebecca Tanous2, Dominis A. Arris3. (1) Biology, University of Southern Maine, Portland, Maine, United States (2) University of Southern Maine, Portland, Maine, United States

The purpose of this research is to formulate and test a carbohydrate based additive which can be added to brine solutions to increase ice melting efficacy and adherence to roads. This additive will be extracted from potatoes (low-grade ones unavailable at the grocery store) through a starch to fermentable sugar conversion. This molasses-like substance will be used to treat sodium chloride in both solid and liquid brine solution form. Freezing point will be determined in the lab, and efficacy of mixtures will be determined at varying concentrations. With the state of Maine having 23,450 miles of public roadway (more miles per person than any other N.E. state), an average 10 year annual cost of 1.5 billion dollars spent on road salt, and approximately 490,000 tons of salt dropped statewide per year, there are economic and ecological concerns in lowering the amount of salt applied to the roads. During the winter we will acquire small spaces of asphalt on campus where we can spread the solution during the winter months to help spread awareness about salting roads as well as the University of Southern Maine Chemistry Club and some of the things we’re doing this year.

CARB 55

Amide-linked RNA: Synthesis, structure, and RNA interference activity

Eriks Rozners1, erozners@binghamton.edu, Daniel Mutisya1, Chelliah Selvam1, Paul Tanui1, Benjamin D. Lunstad2, Scott D. Kennedy3, Pradeep S. Pallan4, Amanda Haas2, Devin Leake2, Martin Egli4. (1) Department of Chemistry, Binghamton University, Binghamton, New York, United States (2) Dharmacon, Lafayette, Colorado, United States (3) University of Rochester, Rochester, New York, United States (4) Vanderbilt University, Nashville, Tennessee, United States

Discovery of RNA interference (RNAi) has reinvigorated interest in chemical modifications of RNA for in vivo applications. Our current work is focused on the development of novel nonionic analogues of RNA that have the phosphodiesters replaced by amide linkages (AM1). We hypothesize that reduced negative charge and hydrophobic nature of such modifications will not only increase the enzymatic stability but also have the potential to optimize potency, cellular uptake, and pharmacokinetics of small interfering RNAs (siRNAs). A further advantage of amide modifications is that they can be prepared using relatively straightforward peptide type couplings. This presentation will discuss synthesis, structure and RNAi activity of amide-modified RNA. NMR spectroscopy (1) and x-ray crystallography (2) show that amides are excellent mimics of the phosphodiester inter-nucleoside linkages in RNA and suggest interesting insights into how the formally hydrophobic modification interacts with hydration of RNA. RNAi assays show that multiple amides are tolerated at internal positions in both strands of siRNAs (1,2). Surprisingly, amide modifications at the 5´-end of the passenger strand increased the RNAi activity compared to unmodified siRNA (2). The implication of these studies for improving the properties of siRNAs for potential therapeutic applications will be discussed. References:

(1) Mutisya, D.; Selvam, C.; Lunstad, B. D.; Pallan, P. S.; Haas, A.; Leake, D.; Egli, M.; Rozners, E., Amides Are Excellent Mimics of Phosphate Internucleoside Linkages and Are Well Tolerated in Short Interfering RNAs. Nucleic Acid Res. 2014, 42, 6542–6551. (2) Tanui, P., Kennedy, S.D., Lunstad, B.D., Haas, A., Leake, D. and Rozners, E. Synthesis, biophysical studies and RNA interference activity of RNA having three consecutive amide linkages. Org. Biomol. Chem. 2014, 12, 1207-1210.

CARB 56

Modification of glucose for targeted cellular delivery

Harvey Jacobs2, Zbigniew J. Witczak1, zbigniew.witczak@wilkes.edu. (1) Dept of Pharm SCI Sch of Phar, Wilkes Univ, Wilkes Barre, Pennsylvania, United States (2) Wilkes University, Wilkes Barre, Pennsylvania, United States

Previous research involved targeting polymeric beads coupled with anti -H. pylori antibodies to H. pylori, cells. The immediate reaction of this system was complete agglutination of cells through the through the mulit-point attachment to the beads. One drawback of this system would be the instability of the protein-based antibody in the acidic and enzymatic conditions of the GI tract leading to denaturation and cleavage of the protein resulting in poor binding with the cells. Many studies (1,2) demonstrated the specific binding of carbohydrate moieties to cells when coupled to a polymeric backbone. Furthermore, the specificity of carbohydrate coupling through C-1 or C-6 position may influence cell binding affinities (3,4). The preliminary focus of this research is to modify glucose with vinyl adducts through the C-6 position, leaving the C-1 position available for cell receptor binding. 1) Kobayashi, K;. Sumitomo, H; Ina Y. Polymer Journal, 1983, 15, 9 667-671. 2) Kobayashi, K;. Sumitomo, Macromolecules, 1983, 15, 9 234-239. 3) Tamai, I; Tsuji, A, Advanced Drug Delivery Reviews, 1996 5-20.234-239. 4) Thorens, B. Annu. Rev. Physiol. 1993, 55, 591-608.

CARB 57

Targeted study of bacterial glycoproteins using metabolic oligosaccharide engineering

Danielle H. Dube, Emily Clark, eclark@bowdoin.edu. Chemistry & Biochemistry, Bowdoin College, Brunswick, Maine, United States

Many species of pathogenic bacteria are developing resistance to antibiotic treatments. As such, there is a rising need for novel therapeutic targets. The glycosylated, or sugar-modified, proteins present on bacterial cell surfaces provide one such target. Importantly, glycosylation patterns vary between bacterial and mammalian species, as well as among different bacterial species. This variation creates the potential to selectively target a bacterial species based on cell-surface glycans, while leaving other cells unharmed. The Dube lab has exploited these observations using a process termed metabolic oligosaccharide engineering, in which an unnatural sugar is incorporated into cellular glycans for visualization and targeting purposes. In this project, we explored the extent to which a panel of unnatural sugars is incorporated into glycoproteins in a range of bacterial (e.g. Helicobacter pylori, Campylobacter jejuni, Burkholderia thailandensis, and Bacteroides fragilis) and mammalian cells. Our preliminary results suggest that differential incorporation of azidosugars is possible, such that azides are incorporated onto the surface of select bacteria yet are absent from the surface of mammalian cells and other bacteria. Ultimately, this project has the potential to expand our knowledge of bacterial glycoproteins while identifying optimal metabolic substrates for antibiotic treatment.

CARB 58

Development of a cyclooctyne-based photodynamic antibiotic for targeting Helicobacter pylori's surface sugars

Danielle H. Dube, Ian Kline, ikline@bowdoin.edu. Chemistry & Biochemistry, Bowdoin College, Brunswick, Maine, United States

Helicobacter pylori infects over 50% of people worldwide, causing gastric ulcers and even gastric cancer in some of these individuals. Due to the recent increase of antibiotic resistant strains of H. pylori, existing triple antibiotic treatments for H. pylori are becoming ineffective. We propose an antibiotic that will increase H. pylori's innate sensitivity to light by incorporating the photosensitizer protoporphyrin IX (PpIX) onto the cell membrane. Our two-step therapeutic approach involves first selectively labeling H. pylori’s surface sugars with azides using metabolic oligosaccharide engineering, which has been previously executed by the Dube lab. We then plan to covalently attach the PpIX to the H. pylori cell surface via the bioorthogonal strain-promoted copper free click chemistry reaction between cyclooctynes and azides. To realize this vision, an antibiotic was designed that contained three components: PpIX for photodynamic therapy, aza-dibenzocyclooctyne for reacting with azide-labeled H. pylori, and a C-FLAG peptide to increase hydrophilicity. The desired compound was synthesized first via Fmoc-based solid phase peptide synthesis followed by the coupling of the cyclooctyne onto the peptide-PpIX complex using thiol-maleimide chemistry. Liquid chromatography-mass spectrometry confirmed the successful synthesis of the final compound, DIBAC-Cys(PpIX)-FLAG. The purified product was obtained using high performance liquid chromatography. Western blotting confirms that the compound can bind to azide-labeled surface glycans on H. pylori. Experiments are currently underway to assess the ability of this compound to catalyze light-dependent killing of azide-covered H. pylori. Ultimately, this work has the potential to treat patients with antibiotic resistant strains of H. pylori with a minimally invasive endoscopic procedure that will deliver light to photosensitized H. pylori.

CARB 59

Analysis of Helicobacter pylori strains deficient in protein glycosylation

Danielle H. Dube, Sage Mikami, smikami@bowdoin.edu, Hallie Carol. Chemistry & Biochemistry, Bowdoin College, Brunswick, Maine, United States

Helicobacter pylori is a Gram-negative bacteria that can lead to the development of gastric and duodenal ulcers, gastritis, and gastric cancer in infected individuals. H. pylori is growing increasingly resistant to current antibiotic treatments; therefore a novel approach for treating H. pylori is essential. H. pylori’s ability to sugar-modify, or glycosylate, its proteins is imperative for the function of many proteins involved in H. pylori pathogenesis; therefore, the protein glycosylation pathway is a good candidate for future drug targeting. Our lab has identified several mutant strains of H. pylori deficient in protein glycosylation. Interestingly, some of the identified putative H. pylori protein glycosylation genes are implicated in glycosylating lipids to produce lipopolysaccharide (LPS), a class of molecules that is crucial for H. pylori’s pathogenesis. Here we report our investigation of the potential overlap in protein glycosylation and LPS biosynthesis in H. pylori. In particular, we have analyzed the differences in glycosylation fingerprints

of H. pylori mutant versus complemented strains and of LPS-enriched versus LPS depleted samples. Ultimately, this work will reveal genes involved in H. pylori’s protein glycosylation, which has the potential to reveal novel drug targets for H. pylori.

CARB 60

Synthesis of chitosan beads enclosed magnetic Fe3O4 nanoparticles endorsed several applications from environmental remediation to bio-nanocomposite integration in biomedicine interdisciplinary fields

Victor Fernandez-Alos1, victor.fernandez4@upr.edu, Abigail Padilla1, Catherine Castro-Alvarado2, catherine.castro@upr.edu, Felix R. Roman1. (1) Chemistry, University of Puerto Rico-Mayaguez, Mayaguez, Puerto Rico, United States (2) Chemical Engineer, University of Puerto Rico-Mayaguez, Mayaguez, Puerto Rico, United States

Synergistic effects between the synthesis of magnetite Fe3O4 nanoparticles with chitosan protonated/non-protonated beads can be used in multiple fields such as environmental remediation, drug targeted-delivery systems, magnetic resonance image enhance, and therapeutic administrations among others. Thus, the objective in this investigation was to design simple automated process for production of chitosan/Fe3O4 beads. In an effort to test the synthesis model methodology, different forms of chitosan surface functionality were selected from structure-behavior, environmental cost-effective to energy related processes (e.g. wastewater treatment plants). Trace level of endocrine disrupting compounds (EDCs) on effluent discharged from wastewater treatment plant to environmental water systems had been observed. EDCs are defined as emerging contaminants, which are subdivided on steroid hormones, pesticides, polycyclic aromatic hydrocarbon (combustion), and pharmaceutical and personal care products. According to literatures estrogenic-EDCs with high estrogenic-activity in water systems were obtained from estrogen, 17β-estradiol, and 17α-ethynylestradiol. Integrated bionanocomposite such as chitosan/Fe3O4 magnetic beads with conventional wastewater treatment techniques for the removal and degradation of EDCs specifically steroid endocrine hormones was evaluated in this research project. The magnetic properties enable the recovery of chitosan/Fe3O4 bead adsorbents, thus reducing operational cost in treatment water facilities. Furthermore, cutback on the use of expensive no-reusable adsorbents endorsed several advantages of chitosan magnetic beads for remediation practices.

Synthesis of chitosan/Fe3O4 magnetic beads.

CARB 61

Hybrid ceramide–gypsogenin triterpene saponin as a vaccine adjuvant

Vibha Pathak, pathakv@southernresearch.org, Ashish K. Pathak. Organic Chemistry Department, Southern Research Institute, Birmingham, Alabama, United States

Adjuvants are heterogeneous group of compounds that are capable of triggering or enhancing an immune response against an otherwise poorly immunogenic entity. Gypsohilla saponins are a heterogenic mixture of glycosides that have a gypsogenin triterpene aglycone that carryies an aldehyde group which can stimulate both cellular and humoral immune responses. One of the most active triterpene saponin is QS-21 which bears a tertiary aldehyde. QS-21 is being evaluated as an adjuvant in several cancer, bacterial and viral vaccine preparations. The inherent unstability and toxicity of QS-21 made its clinical use very limited. Based on the structure-activity relationship (SAR) data for the QS-21 and similar saponins, we have designed and tested a unique gypsogenin–ceramide saponin hybrid. This hybrid molecule shall synergestically activate natural killer T (NKT) cells along with other Th1 and Th2 immune stimulating adjuvant activity in mice with recombinant hemagglutinin of influenza A (rHA) antigen. The progress of the design, the synthesis and the activity on this new Gypsogenin saponin will be presented.

CARB 62

Cell-surface engineering with biomimetic materials: Mucins and the cancer glycocalyx

Jessica Kramer1, jessk713@chem.ucla.edu, Carolyn R. Bertozzi2. (1) UC Berkeley, Oakland, California, United States (2) Univ of California, Berkeley, California, United States

Cell-surface glycans play essential roles in diverse cellular interactions. An illustrative example is the membrane associated mucin glycoprotein MUC1, which is highly over-expressed and aberrantly glycosylated on ca. 90% of epithelial carcinomas and is a known cancer biomarker. However, correlation of glycan structure to function in glycobiology is complicated by the varied and dynamic nature of glycan presentation and a current lack of biochemical techniques to precisely modify and identify glycans. We have established a versatile chemical route to prepare a library of well-defined, fully synthetic glycopolypeptide biomimics of the mucin extracellular glycodomain. Using this library, we have identified unique biophysical and morphological properties of mucin glycosylation. To characterize glycan function, we next use cell-surface engineering to display the biomimetic glycopeptide library on the surface of live cells via lipid anchors or engineered protein anchors. Currently, we are investigating the functional significance of how subtle changes in mucin glycosylation and expression levels influence downstream effects on cancer related cellular adhesion, survival, and migration.

CARB 63

Structural characterization of a newly discovered trisaccharide in banana fruit ethanol extract

Michael A. Madson, mmadson53@earthlink.net. Research & Development, BioLogistics LLC, Ames, Iowa, United States

One of the possible structures for these ions is fructosyl sucrosyl-1,6-di-phosphate, with the two phosphate groups located at the two primary positions of the fructo-furanoside of the sucrose of the trisaccharide. This compound may be an effective lead for the inhibition of glycolysis, at elevated levels in cancer, at the point of conversion of fructose-1,6-bisphosphate to di-hydroxy acetone phosphate and glyceraldehyde-3-phosphate with fructose-1,6-bisphosphate aldolase. Also glycolytic aldolase A and C have been shown to bind to NF-L, neutrofilament L, mRNA that is a portion of mRNA of the total NF-L mRNA important in preventing neuro-degenerative disease. Aldolase is elevated in amyotropic lateral schlerosis (ALS) and over-expressed NF-L, in a mouse model, delays onset of ALS. Thus inhibitors, possibly the compound from ethanol extract from banana, of this enzyme may be of help in treating ALS. Alzheimers Disease aldolase is know to be present in AD patients and an antibody to it causes a lowering of AD symptoms in mice. Inhibition of this enzyme may be a pathway to the inhibition of AD symptoms in humans. Aldolase in Mycobacterium tuberculosi is known to be important in its metabolism. Inhibitors of its aldolase may be an effectiive treatment for this microbe. We have discovered a novel trisaccharide from an ethanol extract of banana fruit. We have done ICP-MS and ICP-MS/MS of the molecule in negative mode. We allowed a prepared ethanol extract of banana fruit to stand for 2 weeks or less before we did ICP-MS in the negative ion mode. The positive mode indicates the presence of sucrose, fructose and di-phospho fructosyl sucrose. The negative mode ICP-MS indicates intact di-phosphoryl fructosyl sucrose and its cleavage ions. We also present negative ion mode ICP-MS from a portion of the banana fruit extract treated diluted with ~1N ammonium hydroxide and to this solution sodium borohydride. This solution is allowed to stand for more than 1 day. After more than one day at these conditions the solution is diluted with ethanol and submitted for analysis by an ICP-MS instrument. We have evidence of hydride inserted fructosyl di-phospho fructosyl sucrose molecular ion, m/z 719.209 and molecular ion, m/2, 376.79. We will present their possible structure along with the accuracy of calculated versus found atomic mass units for the proposed ions for identity confirmation.

CARB 64

Effect of conjugation and microwave treatment on structure and functional characteristics of gum karaya (Sterculia urens)

Hamed Mirhosseini, hamedmi@upm.edu.my, Elhamalsadat Shekarforoush. Universiti Putra malaysia, Serdang, Se, Malaysia

In the last decade, the customer demand for natural gums from plant sources has been extensively increased. However, some of plant gums have several technical problems such as low solubility and low particle uniformity. Karaya (Sterculia urens) plant gum with many functional properties mainly used in cosmetic and pharmaceutical products rather than food products. This may be due to its strong swelling properties and weak solubility. In this research, we investigated the effect of gum karaya when is mixed or conjugated with whey protein isolate (WPI) or soy protein isolate (SPI), besides gum

karaya to microwave irradiation at 700 W or 1000 W for 8, 10, or 12 min to compared with gum karaya (control) . The functional characteristics of karaya gum in the aqueous system were observed including the solubility, swelling Index (SI), water holding capacity (WHC), oil holding capacity (OHC) and Fourier transform infrared spectroscopy (FTIR). Fourier transform infrared spectroscopy of conjugated gum karaya confirmed that there was protein–gum covalent linkage. The solubility was altered between 20 to 65% for protein–gum mixtures and conjugated biopolymers. The difference might be explained by the ratio of gum karaya to protein. On the other hand, there were physical changes after microwave irradiation. FTIR spectra showed similar peaks and attributes in the control and microwave irradiated gum. However, the solubility was 17–47% compared to 22.5% in the control. This was dependent upon the power–time microwave parameters. The swelling ratio was the lowest in the heat-treated gum solution microwave treated at 700 W for 8 min between all microwaves irradiated and control samples. It probably affected the water–compound interaction by reducing the number of areas capable of active hydration.

CARB 65

Synthesis, characterization, and application of soy protein flour-based additive to increase the dry strength of recycled and virgin paper furnish

Abdus Salam, masalam@ncsu.edu, Lucian Lucia, Hasan Jameel. Forest Biomaterials, NC State University, Raleigh, North Carolina, United States

Soy protein flour as a potential new comer to the field is a modestly priced, yet complex glycoprotein-based biomacromolecule compared to a number of other paper dry strength additive. Nevertheless, and perhaps more importantly, it possesses a relatively rich hydrogen-bonding surface functional density, but high susceptibility to bacterial digestion due to its protein-based composition. Unfortunately, within the construct of any commercial paper-based applications, the results of the digestion are a characteristically unpleasant odor, machine fouling, and potential paper strength losses, vital issues to consider for its potential application as a dry strength additive. Soy protein flour was modified with diethylenetriaminetetracaetic acid and further crosslinked with chitosan. The synthesis conditions were optimized. The tensile indices of modified soy protein flour additive-treated recycled OCC pulp sheets, NSSC (virgin) pulp sheets, and kraft (virgin) pulp sheets increased by 52%, 53%, and 58%, respectively, while the inter-fiber bonding strength increased 2.5-3.0 times. Decomposition of soy protein flour was studied under open-air conditions. The unmodified soy protein flour decomposed within 24 hours as indicated by its characteristically foul odor, an observation that did not hold for the modified soy protein flour nearly two years. Antimicrobial activity was tested. Soy protein flour significantly increased bacteria growth compare to control (blank). But modified soy protein flour was killed 100% bacteria. OCC pulp sheet was not shown any antimicrobial activity but modified soy protein flour-treated sheet was killed about 93-97% bacteria compare to pulp sheet.

CARB 66

Structural insight into glycosylated human Notch1 EGF12 analogs

Shun Hayakawa, shun.hy@mail.sci.hokudai.ac.jp, Hiroshi Hinou, Shin-Ichiro Nishimura. Grad Sch of Life Sci, Hokkaido Univ, Sapporo, Japan

Notch signaling pathway plays key roles of cell developing process. Notch protein contains 36 epidermal growth factor (EGF)-like repeats on extra cellular domain. Each EGF domains have conserved three disulfide bonds. Many EGF domains are glycosylated by O-fucose [mature form is Neu5Acα(2→3)Galβ(1→4)GlcNAcβ(1→3)Fucα1→Thr] and O-glucose [Xylα(1→3)Xylα(1→3)Glcβ1→Ser]. O-Fucosylation of EGF12 is one of the most important modification to Notch signaling activities because glycosylation pattern of this

O-glycan regulates binding affinity with their ligands[1]. However, the detail functions of O-fucosylation as well as O-glucosylation are still unclear. Previously, we reported chemical synthesis and NMR structural analysis of mouse EGF12 peptide carrying O-fucosyl glycans, and demonstrated that local peptide conformation altered by glycan elongations [2]. In the present study, our attention is focused on human EGF12 and impact of O-glucosylation in addition to O-fucosylation. Here, we present chemical synthesis of human Notch1 EGF12 glycopeptides carrying O-fucose- or O-glucose-type glycans. In the folding reaction of hEGF12 analogues, we revealed that folding reaction proceeded efficiently in the presence of calcium ion in redox buffer. These synthesized EGF12 glycopeptide analogues are subjected to NMR conformational study. [1] P. A. Handford et. al. Proc. Natl. Acad. Sci. U.S.A. 2014, 20, 7290-7295. [2] S.-I. Nishimura et. al., J. Am. Chem. Soc. 2010, 132, 14857-14865.

Synthetic glycosylated human Notch1 EGF12 analogues

CARB 67

Metabolism of four metabolic chemical reporters and their relative selectivity for different glycoproteins

Anna Batt1, Annabatt@gmail.com, Matthew Pratt2. (1) Chemistry, University of Southern California, Los Angeles, California, United States (2) University of Southern California, Los Angeles, California, United States

Protein glycosylation is one of the most abundant and complex forms of posttranslational modification in mammalian cells. Metabolic chemical reporters (MCRs)

have been indispensable for the visualization and identification of glycoproteins. Typically, MCRs are monosaccharide analogs that contain small bioorthogonal functional groups that are tolerated by cellular metabolism and become incorporated into glycoproteins. These functional groups can then be labeled using reactions, such as the copper(I) catalyzed azide-alkyne cycloaddition (CuAAC). Recently, we and others have demonstrated that most MCRs are not specific for one type of glycosylation. However, structural changes to the MCR could bias their incorporation into different glycans. Here I will present data on the metabolism of four different MCRs that explains their relative selectivity for different types of glycoproteins, with important implications for their application by the glycoprotein community.

CARB 68

Changes in metabolic chemical reporter structure yield a selective probe of O-GlcNAc modification

Kelly N. Chuh, kchuh@usc.edu. Chemistry, University of Southern California, Los Angeles, California, United States

Metabolic chemical reporters (MCRs) of glycosylation are analogues of monosaccharides that contain bioorthogonal functionalities and enable the direct visualization and identification of glycoproteins from living cells. Each MCR was initially thought to report on specific types of glycosylation. We and others have demonstrated that several MCRs are metabolically transformed and enter multiple glycosylation pathways. Therefore, the development of selective MCRs remains a key unmet goal. We demonstrate here that 6-azido-6-deoxy-N-acetyl-glucosamine (6AzGlcNAc) is a specific MCR for O-GlcNAcylated proteins. Biochemical analysis and comparative proteomics with 6AzGlcNAc, N-azidoacetyl-glucosamine (GlcNAz), and N-azidoacetyl-galactosamine (GalNAz) revealed that 6AzGlcNAc exclusively labels intracellular proteins, while GlcNAz and GalNAz are incorporated into a combination of intracellular and extracellular/lumenal glycoproteins. Notably, 6AzGlcNAc cannot be biosynthetically transformed into the corresponding UDP sugar-donor by the canonical salvage-pathway that requires phosphorylation at the 6-hydroxyl. In vitro experiments showed that 6AzGlcNAc can bypass this roadblock through direct phosphorylation of its 1-hydroxyl by the enzyme phosphoacetylglucosamine mutase (AGM1). Taken together, 6AzGlcNAc enables the specific analysis of O-GlcNAcylated proteins, and these results suggest that specific MCRs for other types of glycosylation can be developed. Additionally, our data demonstrate that cells are equipped with a somewhat unappreciated metabolic flexibility with important implications for the biosynthesis of natural and unnatural carbohydrates.

CARB 69

Efficient method for the incorporation of molecular probes at multiple/specific sites in RNA: Levulinyl protection for 2’-ACE®, 5’-silyl oligoribonucleotide synthesis

Michael O. Delaney, michael.delaney@thermofisher.com. GE Healthcare Dharmacon, Inc., Lafayette, Colorado, United States

Molecular probes have found wide application in the study of biomolecules within living systems. Oligonucleotides that are labeled with molecular probes are an invaluable tool for monitoring DNA and RNA processing for both in vitro and in vivo applications. Solid-phase oligonucleotide synthesis facilitates relatively straightforward and efficient incorporation of molecular probes at the 5’ end of DNA or RNA. However, modifying the 3’-end of an oligonucleotide generally requires either post-synthetic strategies or immobilization of the molecular probe to the solid support. The former process is subject to low yields due to potentially inefficient coupling while the latter strategy is restricted by the stability of the modification to repeated exposure to synthesis reagents. Similarly, internal labeling of oligonucleotides with molecular probes is largely limited to post-synthetic processing and subject to coupling efficiencies associated with this process for these labeling steps. Finally, the need to differentially label oligonucleotides with distinct moieties in specific terminal and internal positions adds yet another layer of complexity in the generation of these important molecular tools. In order to improve the labeling efficiency and ease of preparation of internal or 3’-terminal sites of oligoribonucleotides, we have developed a method for labeling these positions while the oligonucleotide remains immobilized on the solid support. We have applied a method to selectively de-block a levulinyl-protected hydroxyl group at a variety of different sites within an oligonucleotide and to selectively label these positions by the use of phosphoramidite-activated molecular probes. Conditions used to remove the levulinyl protecting group are mild and compatible with the 2’-ACE®, 5’-Silyl oligoribonucleotide synthesis platform, resulting in excellent yields of high quality, full length modified oligoribonucleotide. Examples will be given demonstrating the utility of this methodology to efficiently label internal positions and at the 3’-end of an oligonucleotide. Furthermore, applications of this methodology for introducing multiple molecular probes at defined positions within oligonucleotide will be presented.

CARB 70

Aldehyde bearing triterpene saponins as vaccine adjuvants

Vibha Pathak, Ashish K. Pathak, pathaka@sri.org. Organic Chemistry Department, Southern Research Institute, Birmingham, Alabama, United States

Commercially available Quillaja and Gypsophilla saponin extracts are rich in aldehyde-containing triterpene saponins and exhibit Th1 and Th2 immune-stimulating activities as adjuvants. One of the most active saponins is known as QS-21 which is isolated from Q. saponaria. QS-21 is currently being evaluated as an adjuvant in several cancer, bacterial and viral vaccine preparations. However, the clinical use of QS-21 is limited due to its low stability in solution at physiological pH and its high toxicity at doses required for maximum immune adjuvanticity. Several synthetic analogs, such as GPI-0100 and semi-synthetic QS21 analogs, are currently being evaluated as adjuvants.

The design, synthesis and structure/activity relationships of a new series of semi-synthetic saponin analogs of Quillaic acid and Gypsogenin will be discussed. These new saponin analogs show no dose dependent toxicity up to 4 mg/injection and exhibit excellent immune-stimulating activity in mice with recombinant hemagglutinin of influenza A (rHA) antigen.

CARB 71

Xanthan: Conformation, degradation, and hydrophobic modification

Ina Beate Jenssen1, Ann-Sissel Ulset1, Henk A. Schols2, Audrey Roy3, Frédéric Renou3, Bjoern E. Christensen1, b.christensen@biotech.ntnu.no. (1) Biotechnology, NTNU, Trondheim, Norway (2) Food Chemistry, WU Agrotechnology & Food Sciences, Wageningen, Netherlands (3) †University of Le Havre, Le Havre, France

The industrial polysaccharide xanthan is produced by fermentation and used as a viscosifier in a variety of applications, ranging from food additives to oil recovery1. The high intrinsic viscosity is a result of extremely high molecular weights and the stiff, double-stranded conformation. The latter also provides a ‘DNA-like’ behaviour upon degradation, and provides shear thinning and salt-insensitive solution viscosity. Xanthan and pyruvate/acetate-free xanthan were both degraded by ultrasonic treatment and by the Fenton reagent2,3 The fragments were analysed by multi-detector SEC, providing the classical [h]-M and RG-M relationships, and compared to corresponding data obtained by static light scattering (literature data)4,5. Whereas sonication tended to produce essentially double-stranded fragments, the free radical induced degradation gives rise to multimodal distributions6 and metastable structures7 Sonicated xanthans were further modified8 by adding octylamine substituents (Degree of substitution, DS = 0.06 – 0.20) while retaining the ordered conformation as shown by multi-detector SEC using a modified solvent.

The purpose of this study is to evaluate the influence of substitution and DS on the degradability by cellulase for xanthan chains of different molecular weights. Preliminary results will be presented. (1) Stokke, B. T.; Christensen, B. E.; Smidsrød, O. In Polysaccharides . Structural diversity and functional versatility.; Dumitriu, S., Ed.; Marcel Dekker Inc.: New York, 1998, p 433. (2) Hjerde, T.; Kristiansen, T. S.; Stokke, B. T.; Smidsrød, O.; Christensen, B. E. Carbohyd Polym 1994, 24, 265. (3) Christensen, B. E.; Myhr, M. H.; Smidsrød, O. Carbohyd Res 1996, 280, 85. (4) Sato, T.; Norisuye, T.; Fujita, H. Macromolecules 1984, 17, 2696. (5) Matsuda, Y.; Biyajima, Y.; Sato, T. Polym J 2009, 41, 526. (6) Christensen, B. E.; Smidsrød, O.; Elgsaeter, A.; Stokke, B. T. Macromolecules 1993, 26, 6111. (7) Christensen, B. E.; Smidsrød, O.; Stokke, B. T. Macromolecules 1996, 29, 2939. (8) Roy, A.; Comesse, S.; Grisel, M.; Hucher, N.; Souguir, Z.; Renou, F. Biomacromolecules 2014, 15, 1160.

CARB 72

Alkali pretreatment of cellulose I to cellulose II with thiourea as an additive

Vanshi Uniyal2, vanshi.uniyal@gmail.com, P. K. Gupta2, Sanjay Naithani1. (1) Chemistry of Forest Products Division, Institute of Wood Science and Technology, Malleswaram, Bengaluru, India (2) Centre for Advanced Studies in Chemistry of Forest Products, Forest Research Institute, Dehra Dun, India

The effect of thiourea as an additive on the crystalline structure of cellulose was studied by treating it with different concentrations of sodium hydroxide. The alkali treated cotton linters with and without thiourea as an additive were analyzed by wide-angle X-ray diffraction analysis. The structural transformation of cellulose from cellulose I to cellulose II was observed at 15wt% concentration of sodium hydroxide. The crystallinity of the treated cotton linter declined gradually till 10wt% concentration of sodium hydroxide and at 15wt% concentration of sodium hydroxide was reduced largely with or without the addition of thiourea. The crystallinity index showed a gradual decrease with increasing concentration of sodium hydroxide with or without thiourea as an additive. However, the addition of thiourea at 15wt% concentration of sodium hydroxide had a great impact on lowering the crystallinity of the cellulose.

CARB 73

Immunomodulation of the linear b-(1,3)-glucan frSaccharomyces cerevisiae through activation of mitogen-activated protein kinases and nuclear factor-kB in murine RAW264.7 macrophages

Xiaojuan Xu, xuxj@whu.edu.cn. Wuhan University, Wuhan, China

BBG, a linear b-(1,3)-glucan from Saccharomyces cerevisiae (Baker’s yeast), is a major component of yeast cell wall, showing versatile physiological activities, such as anti-tumor, anti-viral, anti-inflammatory, immunoregulation and so on. As we known, pattern recognition receptors (PRRs) play critical roles in these reactions. Dectin-1 and CR3 are major receptors of b-glucans. In this study, we reported that BBG activates murine RAW264.7 macrophages and induces production of cytokines including TNF-a, IL-10, MIP-2 and MCP-1, leading to immune responses. Nuclear factor-kB p65 and its nuclear translocation were determined by western blotting. The upstream signaling molecules, MAP kinases such as JNK1/2 and ERK1/2, were confirmed to be activated by assessing the level of phosphorylation in a time-dependent manner, while the downstream proinflammatory factor NO was not detectable. Further studies confirmed that BBG activated RAW 264.7 macrophages to produce TNF-a and MCP-1 through activating MAP family kinases of JNK1/2 and ERK1/2, and promoting nuclear translocation of nuclear transcription factor NF-kB p65. However, dectin-1 was not detected on RAW 264.7 macrophages while CR3 and TLR-2 were. This suggested that BBG activate the production of cytokines through a dectin-1-independent pathway in RAW 264.7 macrophages. Moreover, CR3 and TLR-2 were involved in BBG-induced TNF-a and MCP-1 production. All of the results demonstrated that BBG carries out its immunomodulatory activity by activating MAPKs/NF-kB signaling pathways.

CARB 74

O-mannosylated glycan induced conformational alteration of α-dystroglycan fragment

Hiroshi Hinou, hinou@sci.hokudai.ac.jp, Shin-Ichiro Nishimura. Graduate School of Life Science, Hokkaido University, Hokkaido, Japan

Muscular dystrophy (MD) is caused by genetic disorder to progresses muscle atrophy and muscle weakness. Endo et al. reported that defect of protein O-Mannosyltransferase (POMT1, 2) or protein O-mannose β1,2-N-acetylglucosaminyltransferase (POMGnT1) are cause the symptoms of MD.1) Recently, Campbell et al. reported that phosphorylated O-mannosyl glycan is required for the function of dystroglycan, a cell surface protein of neural and muscular cells, to bind with extracellular protein, laminin.2) However, POMGnT1, the MD related glycosyltransferase, is not involved in the phosphorylated glycan synthesis, and no rational function of this enzyme is still unrevealed. To elucidate the function of the GlcNAc residue transferred by POMGnT1, we constructed a glycopeptide library of the mucin-like domain of human a-dystroglycan bearing POMGnT1 modified-type O-mannosylated glycans at Thr residue. The glycopeptides displayed microarray (Sumitomo Bakelite Co.,Ltd.) shows week interaction of a glycopeptide with laminin. Conformational study of the selected glycopeptides by NMR showed that the introduction of a GlcNAc residue induce conformational changes of the peptide backbone. This result indicates that the POMGnT1 works as conformational modulator

to activate further glycan modifications. 1) T. Endo et al. J. Biol. Chem. 274, 2156-62 (1997); Developmental Cell 1, 717-24 (2001). 2) Takako Yoshida-Moriguchi et al. Science 327, 88 (2010); Science 341, 896 (2013).

CARB 75

Efficient and α-selective glycosylation using 3-iodo Kdo (3-deoxy-D-manno-oct-2-ulosonic acid) fluoride donors

Barbara Pokorny, barbara.pokorny@boku.ac.at, Paul Kosma. Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria

In the Gram-negative bacterial cell wall α-Kdo is an important component of the inner core region of lipopolysaccharide (LPS), which is frequently recognized by lectins and antibodies of the immune system in hosts.[1] Chemical glycosylation using Kdo donors, however, is typically hampered by the lack of a stereodirecting group at C-3, their propensity towards elimination reactions and the low intrinsic reactivity due to the deactivating C-1 ester. In this contribution, the application of easily accessible 3-iodo Kdo fluoride donors (Scheme 1) on the efficient assembly of LPS fragments of the Chlamydiaceae family and the Acinetobacter species – the latter one being responsible for severe nosocomical infections – will be presented. Notably, these glycosylations are highly α-selective and elimination reactions are largely suppressed affording good to high isolated yields (58 to 83%) of the exclusively α-linked products. Capitalizing on a mild hydrogen atom transfer reaction[2] the auxiliary group is subsequently removed cleanly. Sequential iteration of the basic concept allows for linear oligosaccharide synthesis. [1] Weis W.I., et al.; Immunol. Rev. 1998, 163, 19 – 34 [2] Boivin J., et al.; Chem. Commun. 1997, 4, 353-354 Financial support from the Austrian Science Fund (FWF) is gratefully acknowledged (P24921-N28).