Research Descriptions

The following are the "Abstracts" and "Specific Aims" of funded research projects underway in my laboratories in the Department of Chemistry and Biochemistry at the University of Notre Dame, and at Omicron Biochemicals Inc. in South Bend. These laboratories collaborate on research projects through the sharing of complementary expertise, equipment and research resources. Both laboratories are independently funded and managed. In the present global research environment, university-industry linkages are now commonly viewed as critical factors in maintaining and enhancing national research competitiveness. The Notre Dame-Omicron synergy has been extremely effective in this role over the past twenty-five years.

One of the positive consequences of this cooperation is the broader training students receive in my laboratory. At Notre Dame, students pursue basic research problems, but also have the opportunity to observe the translation of basic research into practical applications in the commercial workplace. Exposure to a wider range of experimental techniques is also facilitated, since the Notre Dame and Omicron groups use different, yet complementary, scientific methods to reach their research objectives. Both laboratories also engage in off-campus collaborations on a regular basis to secure research expertise not present in the groups and/or to pursue joint research on problems of mutual interest.

Overview, and Statement of Objectives and Methods. Cell-surface oligosaccharides play critical roles in biology. In humans, they are covalently attached to soluble and membrane-bound proteins and lipids, and their biological roles include cell-cell recognition, motility, signal transduction, microbial pathogenesis, and immune recognition. Central to understanding these roles is the contribution from three-dimensional structure and dynamics. Conformational flexibility is common in oligosaccharides due to motions about their glycosidic linkages and in their constituent monosaccharides. Accurate structural interpretations of experimental observables such as those provided by nuclear magnetic resonance (NMR) spectroscopy must take these motions into account. However, present NMR methods of establishing 3D structures of oligosaccharides are deficient in that: (a) they do not allow a reliable quantitative conformational classification of glycosidic linkages; (b) they cannot independently determine the populations of multiple glycosidic linkage conformers in chemical exchange; and (c) data interpretation often relies on input from theoretical calculations such as molecular dynamics (MD) simulations. The deficiency in (c) is serious because MD predictions for saccharides are difficult to validate independently by experiment, and thus the heavy reliance on them to interpret experimental observables is tenuous. The effect of context on glycosidic linkage conformations is also poorly understood at present. Structure-reactivity and structure-function relationships in chemical and biological systems involving oligosaccharides cannot be firmly established until solutions to these problems are found. This study has three primary objectives: (1) Develop new parameterized equations that relate NMR J-couplings in oligosaccharides to specific glycosidic linkage torsions; (2) Measure J-couplings in biologically relevant oligosaccharides and interpret them quantitatively using a new computer algorithm that reveals conformational populations in solution; and (3) Use the results from (2) in comparisons to, and in validations of, MD results obtained on the same oligosaccharides, and in studies of the effects of structural context on linkage conformations.

Intellectual Merit. NMR J-couplings, which are central to 3D structural studies of oligo- and polysaccharides, remain poorly understood. This project aims to fill this knowledge void. Monosaccharides that comprise oligo- and polysaccharides are rich in J-couplings and comparatively devoid of other NMR observables (e.g., inter-residue NOEs) used in structural studies of polypeptides and polynucleotides. Multiple J-couplings across glycosidic linkages can be used to determine the relative orientations of contiguous monosaccharides and, when used with other NMR parameters (e.g., residual dipolar couplings), reveal how the oligosaccharide folds. Structural information encoded in J-couplings can be obtained only if quantitative relationships between their magnitudes and molecular structure have been established. This project will establish these relationships quantitatively and use them to determine oligosaccharide linkage conformational equilibria in solution based solely on experimental data. A deeper understanding of how glycosidic linkage conformations are affected by structural context will be achieved, and new experimental methods to investigate the unique solution behaviors of oligosaccharides (e.g., H-bonding) are anticipated. The development of this J-coupling methodology will promote future conformational studies of glycan conformations in glycoproteins.

Broader Impacts. The methodology developed in this project is applicable not only to structure studies of complex saccharides, but also in regions of proteins and nucleic acids where multiple J-couplings exist (e.g., furanose rings of nucleic acids; side-chains of proteins). In addition, since the experimental methods developed in this project provide a purely experimental approach to assigning oligosaccharide linkage conformational populations in solution, a means of validating conformational predictions obtained from theoretical methods such as molecular dynamics (MD) simulations becomes available. This latter impact is enormous; validation will permit refinement of these theoretical methods, thus leading to their more reliable application, especially in systems where experimental observations are either difficult or impossible to make.

In this Phase I project, a subset of human milk oligosaccharides (HMOs) ranging from disaccharides to pentasaccharides will be prepared on scales ranging from several grams to kilograms. Specifically, N-acetyl-lactosamine will be chemically prepared in kilogram quantities, six HMOs will be chemically prepared on multi-gram scales (10-100 g), and several HMOs will be extended on smaller scales (1-10 g) with N-acetyl-neuraminic acid using a CMP-sialic acid donor and commercially available sialyltransferases. The primary aims of the project are to chemically prepare key HMO building block molecules on large scales, and basic HMO units on modest scales, and to demonstrate the commercial practicality of extending several of the latter basic HMOs to larger sialyl-containing structures via enzymic modification. All syntheses will be conducted under cGLP conditions; existing Omicron documents, procedures, SOPs, methods and laboratory policies developed in prior cGMP projects will be modified for use in this work. For each oligosaccharide product, high-resolution ¹H (600 MHz) and ¹³C (150 MHz) NMR data will be obtained to confirm structure and purity. The NMR characterization will include an effort to assign the ¹H and ¹³C signals as completely as possible, with assistance from 2D homo- and heteronuclear datasets. For each oligosaccharide product, high-resolution mass spectral (HRMS) data will also be obtained to confirm structural assignments made by NMR, and high-performance liquid chromatography (HPLC) will be used to confirm chemical purity. A major tangible outcome of this project will be the identification and implementation of synthetic methods capable of producing small HMOs on large scales for future pre-clinical trials.

PROJECT NARRATIVE:
The surfaces of human cells are coated with tightly bound sugar molecules that play key roles in normal and abnormal metabolic processes such as immunity, cancer and bacterial infection. Knowledge of the three-dimensional shapes of these cell-surface sugars is essential to understanding how sugars bind to cell receptors and mediate metabolic processes, and how this binding might be manipulated or suppressed through therapeutic intervention. This project will develop a new analytical structural method that will elevate and refine current understandings of the biological properties of sugar structure in solution and in the solid state.

PROJECT SUMMARY:

This project will develop a new experimental method known as solid-state nuclear magnetic resonance spectroscopy (ssNMR) to analyze and assign the three-dimensional shapes (conformations) of saccharides (sugars) in solution and in the solid state. The approach is cross-disciplinary, relying on the synthesis of saccharides containing multiple ¹³C isotopes that are introduced at specific carbons, x-ray crystallography to provide precise three-dimensional structures of target saccharides that will be subsequently studied by ssNMR, and calculational tools (density functional theory; DFT) to provide quantitative relationships between saccharide molecular structure and specific NMR parameters known as NMR spin-couplings (J-couplings), which are measured between two ¹³C nuclei in a ¹³C-labeled molecule. The new method will advance current understanding of the shapes of saccharides in solution, which heavily dictate their biological functions in vivo, and provide an urgently needed means to validate theoretical calculations such as molecular dynamics simulations that are commonly used to predict saccharide 3D structures in solution when experimental methods are either unavailable or unable to make these determinations. The primary aims of the project focus heavily on demonstrating the ability of ssNMR to accurately measure specific NMR J-couplings between ¹³C nuclei in ¹³C-labeled saccharides using simple, well-defined systems. The target systems were selected to provide a comprehensive test of the method by investigating multiple conformational features in saccharides, including aldopyranosyl and aldofuranosyl ring, exocyclic hydroxymethyl group, O-glycosidic linkage, and C-O bond conformations. Execution of the research plan will provide a thorough evaluation of the advantages and limitations of the methodology, reliable estimates of measurement errors and sensitivity, and a strong foundation on which to base future applications of the methodology. Longer-term applications include its use to investigate saccharide-receptor complexes, which are difficult to study at the molecular level using present analytical methods, and determinations of oligosaccharide structures on glycoproteins. Given the increasing recognition of saccharides as mediators of key human metabolic processes in vivo such as immunity, cancer and bacterial infection, advances in understanding free and bound conformations of saccharides will promote the discovery of new therapeutics that selectively interfere with undesirable or harmful saccharide molecular interactions in vivo, leading to new or improved treatments or cures of saccharide-based human disease.

PROJECT SPECIFIC AIMS:

Carbohydrates play central roles in biological metabolism, serving as primary energy sources and as functional covalent modifiers of macromolecules such as proteins and lipids. In their latter roles, they serve as mediators of cell-cell recognition and innate immunity, and as anchors of viral and bacterial particles to host cell plasma membranes during infection. Understanding these and other functional roles at the molecular level requires a complete understanding of saccharide solution properties, as free molecules in solution, as ligands bound to cell receptors, and as appendages covalently attached to protein or lipid. These properties are dictated by molecular structure at different levels, from covalent bonding to conformational equilibria and exchange. Unlike many biomolecules, saccharides are highly conformationally flexible, which renders them particularly difficult to investigate by nuclear magnetic resonance (NMR) spectroscopy unless the time-averaged parameters provided by NMR can be interpreted to give information on conformational populations in solution. Once these properties are understood, determining how they change upon binding to receptors or upon attachment to protein are important steps towards designing molecules that inhibit binding (i.e., drug development) or to determining how molecular context (tethering) affects saccharide mobility and thus biological function. Currently it is difficult to (1) quantify the conformational properties of saccharides in solution in terms of populational equilibria, and (2) determine saccharide conformations when bound to biological receptors or tethered to protein, thus impeding progress in structural glycobiology. This project aims to address these problems by investigating and developing a new NMR method based on the novel integration of analytical methods (solid-state NMR and x-ray crystallography), computational methods (density functional theory, DFT), and isotopic synthesis. The successful development of this method will impact not only the field of structural glycobiology, but also related biomedical fields where molecular flexibility impedes or complicates structure-function studies.

The goals of the project are (1) to develop NMR J-couplings as useful structural parameters in solid-state NMR (ssNMR) studies of saccharides; (2) to validate DFT calculations of NMR J-couplings in order to render their use in solution determinations of saccharide conformation more reliable; (3) to establish whether J-couplings measured in saccharide-protein complexes in the solid state can be used to determine bound geometry of the saccharide; and (4) to provide an experimental alternative to investigate ligand-receptor complexes in solids that are not of sufficient quality to permit conventional determinations by x-ray crystallography. If successful, it is imagined that the method could be extended to other important problems in structural biology such as determining side-chain conformation in labeled proteins and oligosaccharide conformations in glycoproteins. These goals will be achieved through pursuit of the following four Specific Aims.

Specific Aim 1. Synthesize, purify and crystallize a group of doubly ¹³C-labeled monosaccharides and disaccharides. Monosaccharides will include methyl aldopyranosides (six-membered rings) and methyl aldofuranosides (five-membered rings), and specific ¹³C-labeling will allow measurements of ¹J_CC, ²J_CCC, ²J_COC, ³J_CCCC and ³J_CCOC in solution and in solid samples. Disaccharides that adopt different O-glycosidic linkage conformations will be studied, and specific ¹³C-labeling will allow J_CC measurements across their internal O-glycosidic linkages (²J_COC and ³J_CCOC values). The available J_CC will be measured in aqueous solution by ¹³C{¹H} NMR spectroscopy to (a) obtain time-averaged values and/or sign information and (b) provide a calibration for subsequent measurements of the same J-couplings in the solid-state.

Specific Aim 2. Obtain high-resolution x-ray crystal structures (if not previously reported) of compounds prepared in Specific Aim 1. Using the same crystals, measure J_CC values by solid-state ¹³C NMR (ssNMR) in the same set of compounds.

Specific Aim 3. Using the x-ray structures obtained in Specific Aim 2, conduct density functional theory (DFT) calculations of the same NMR J_CC values measured in solution (Specific Aim 1) and in the solid-state (Specific Aim 2). Use the solid-state J-coupling measurements obtained on known 3D structures (from the x-ray data obtained in Specific Aim 2) to validate DFT-derived J-coupling-structure correlations used to determine the solution conformational properties of saccharides.

Specific Aim 4. Conduct J-coupling measurements by ¹³C ssNMR in (a) a limited set of labeled oligosaccharides that can be obtained in solid forms but not necessarily single crystalline forms; (b) in a limited set of saccharide-protein complexes that have been crystallized previously. Determine the limitations of the ssNMR method with respect to sample quality (microcrystalline, polycrystalline, amorphous) and magnitude of J-couplings that are measureable in high-molecular weight complexes.