135 results on '"Nicola L. B. Pohl"'
Search Results
2. Automated, Multistep Continuous‐Flow Synthesis of 2,6‐Dideoxy and 3‐Amino‐2,3,6‐trideoxy Monosaccharide Building Blocks
- Author
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Ashley E. DeYong, Alexander Zsikla, Clay S. Bennett, Tu-Anh V Nguyen, Olivea Vasquez, Subbarao Yalamanchili, John Florek, Nicola L. B. Pohl, and Gavin Stamper
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chemistry.chemical_classification ,business.industry ,Continuous flow ,Chemistry ,Monosaccharides ,Amino Sugars ,General Chemistry ,Flow chemistry ,General Medicine ,Modular design ,Automation ,Combinatorial chemistry ,Catalysis ,Article ,chemistry.chemical_compound ,Fully automated ,Deoxy Sugars ,Monosaccharide ,Organic synthesis ,business - Abstract
An automated continuous flow system capable of producing protected deoxy-sugar donors from commercial material is described. Four 2,6-dideoxy and two 3-amino-2,3,6-trideoxy sugars with orthogonal protecting groups were synthesized in 11-32 % overall yields in 74-131.5 minutes of total reaction time. Several of the reactions were able to be concatenated into a continuous process, avoiding the need for chromatographic purification of intermediates. The modular nature of the experimental setup allowed for reaction streams to be split into different lines for the parallel synthesis of multiple donors. Further, the continuous flow processes were fully automated and described through the design of an open-source Python-controlled automation platform.
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- 2021
3. Fully automated fast-flow synthesis of antisense phosphorodiamidate morpholino oligomers
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Genwei Zhang, Chengxi Li, Hong Zong, Nina Hartrampf, David Verhoeven, Carly K. Schissel, Gunnar J. Hanson, Alexander J. Mijalis, Bradley L. Pentelute, Kyle A. Totaro, Nicola L. B. Pohl, Kruttika-Suhas Phadke, Mark D. Simon, Andrei Loas, Ming Zhou, Alex J. Callahan, University of Zurich, and Pentelute, Bradley L
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10120 Department of Chemistry ,0301 basic medicine ,Time Factors ,Morpholino ,Computer science ,Duchenne muscular dystrophy ,Chemistry, Pharmaceutical ,General Physics and Astronomy ,Synthetic chemistry methodology ,Chemistry Techniques, Synthetic ,Communicable Diseases, Emerging ,Morpholinos ,Automation ,0302 clinical medicine ,Solid-phase synthesis ,540 Chemistry ,Chlorocebus aethiops ,Precision Medicine ,Multidisciplinary ,Phosphorodiamidate Morpholino Oligomers ,3100 General Physics and Astronomy ,Nucleic acids ,Fully automated ,RNA, Viral ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,Science ,1600 General Chemistry ,Genetics and Molecular Biology ,Computational biology ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,1300 General Biochemistry, Genetics and Molecular Biology ,medicine ,Animals ,Humans ,RNA, Messenger ,Vero Cells ,Flow chemistry ,Oligonucleotide ,business.industry ,SARS-CoV-2 ,COVID-19 ,General Chemistry ,Oligonucleotides, Antisense ,medicine.disease ,High-Throughput Screening Assays ,COVID-19 Drug Treatment ,Muscular Dystrophy, Duchenne ,Disease Models, Animal ,030104 developmental biology ,General Biochemistry ,Personalized medicine ,business ,030217 neurology & neurosurgery - Abstract
Rapid development of antisense therapies can enable on-demand responses to new viral pathogens and make personalized medicine for genetic diseases practical. Antisense phosphorodiamidate morpholino oligomers (PMOs) are promising candidates to fill such a role, but their challenging synthesis limits their widespread application. To rapidly prototype potential PMO drug candidates, we report a fully automated flow-based oligonucleotide synthesizer. Our optimized synthesis platform reduces coupling times by up to 22-fold compared to previously reported methods. We demonstrate the power of our automated technology with the synthesis of milligram quantities of three candidate therapeutic PMO sequences for an unserved class of Duchenne muscular dystrophy (DMD). To further test our platform, we synthesize a PMO that targets the genomic mRNA of SARS-CoV-2 and demonstrate its antiviral effects. This platform could find broad application not only in designing new SARS-CoV-2 and DMD antisense therapeutics, but also for rapid development of PMO candidates to treat new and emerging diseases., PMOs (phosphorodiamidate morpholino oligomers) have huge potential for antisense therapy but complex and slow synthesis limits application. Here, the authors report the development of automated flow synthesis methods which reduce nucleobase coupling times from hours to minutes removing human errors and allow for high-throughput production.
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- 2021
4. Acid-mediated N-iodosuccinimide-based thioglycoside activation for the automated solution-phase synthesis of α-1,2-linked-rhamnopyranosides
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Alyssa L. Pirinelli, Victoria R. Kohout, and Nicola L. B. Pohl
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010405 organic chemistry ,Chemistry ,General Chemical Engineering ,General Chemistry ,010402 general chemistry ,N-iodosuccinimide ,01 natural sciences ,Combinatorial chemistry ,Solution phase synthesis ,0104 chemical sciences - Abstract
Carbohydrate structures are often complex. Unfortunately, synthesis of the range of sugar combinations precludes the use of a single coupling protocol or set of reagents. Adapting known, reliable bench-chemistry reactions to work via automation will help forward the goal of synthesizing a broad range of glycans. Herein, the preparation of di- and tri-saccharides of alpha 1→2 rhamnan fragments is demonstrated using thioglycoside donors with the development for a solution-phase-based automation platform of commonly used activation conditions using N-iodosuccinimide (NIS) with trimethylsilyl triflate. Byproducts of the glycosylation reaction are shown to be compatible with hydrazine-based deprotection conditions, lending broader functionality to this method as only one fluorous-solid-phase extraction step per coupling/deprotection cycle is required.
- Published
- 2019
5. Development of a Post-Column Liquid Chromatographic Chiral Addition Method for the Separation and Resolution of Common Mammalian Monosaccharides
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Zachary Wooke, Nicola L. B. Pohl, Lauren F. Barnes, and Gabe Nagy
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Mammals ,chemistry.chemical_classification ,Chromatography ,Resolution (mass spectrometry) ,Monosaccharides ,010401 analytical chemistry ,Ligands ,010402 general chemistry ,01 natural sciences ,Mass Spectrometry ,0104 chemical sciences ,Uronic Acids ,chemistry ,Structural Biology ,Animals ,Monosaccharide ,Chromatography, High Pressure Liquid ,Spectroscopy ,Analysis method ,Column (data store) - Abstract
The first solely MS-based methodology for the identification and resolution of the ten common mammalian monosaccharides is presented. Based on Cooks' fixed ligand kinetic method, this technique is effective on multiple classes of monosaccharides and includes the first example of two fixed ligand combinations used in a single multiplexed experiment. Subsequently, a post-HPLC chiral addition method is used in conjunction with this newly developed MS methodology for the separation and identification of mixtures of common neutral mammalian monosaccharides. This proposed technique is able to overcome a limitation of present carbohydrate analysis methods, namely the simultaneous isomeric resolution of multiple monosaccharides in a mixture. Graphical Abstract.
- Published
- 2018
6. Protein N-Glycans: Incorporating Glycochemistry into the Undergraduate Laboratory Curriculum
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Nicola L. B. Pohl, Jill K. Robinson, Andrew G. McKee, Zachary Wooke, Victoria R. Kohout, and Megan C. Thielges
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0301 basic medicine ,Protocol (science) ,Science instruction ,education ,05 social sciences ,050301 education ,Context (language use) ,General Chemistry ,Education ,03 medical and health sciences ,030104 developmental biology ,Mathematics education ,Psychology ,0503 education ,Curriculum - Abstract
Glycoscience continues to be an underrepresented topic in current undergraduate-biochemistry-laboratory curricula. Of the educational laboratories present in this subject area, few introduce students to cutting-edge methods and techniques related to carbohydrates. A multiweek series of experiments is described that highlights a recently published bleach-mediated-carbohydrate-cleavage protocol in the context of the isolation and identification of the soybean glycoprotein, β-conglycinin. Two different levels of undergraduate biochemistry courses (a first-year introductory course and an upper-level bioanalytical course) completed the experimental set. Although the same sequence of experiments took place for both levels, the teaching approaches to the material varied. The introductory course used an inquiry-type learning approach, whereas the upper-level students were taught with a guided-inquiry approach. Students in both courses were able to successfully isolate glycosylated β-conglycinin from inexpensive s...
- Published
- 2018
7. Synthesis of protected glucose derivatives from levoglucosan by development of common carbohydrate protecting group reactions under continuous flow conditions
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Keevan C. Marion, Nicola L. B. Pohl, and Zachary Wooke
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010405 organic chemistry ,Acylation ,Levoglucosan ,Organic Chemistry ,Propanethiol ,Context (language use) ,Chemistry Techniques, Synthetic ,General Medicine ,Carbohydrate ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Article ,0104 chemical sciences ,Analytical Chemistry ,Catalysis ,chemistry.chemical_compound ,Glucose ,chemistry ,Pyridine ,Organic chemistry ,Protecting group - Abstract
Common carbohydrate protecting group reactions under continuous flow processes are reported in the context of producing partially-protected glucose building blocks from levoglucosan. Benzyl ether protection was demonstrated without the use of NaH using barium oxide, which, however, pointed to the need for forms of this catalyst not as susceptible to close packing under flow. Acylation conditions were developed under continuous flow in acetonitrile and avoiding pyridine. Ring-opening the derivatized levoglucosan with propanethiol was also demonstrated producing S-alkyl 2,4-di-O-benzyl-glucopyranoside building block in 2 rather than 12 steps in increased overall yield.
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- 2018
8. Glycobiology of Caenorhabditis elegans
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Shi Yan, Iain B. H. Wilson, Nicola L. B. Pohl, and Katharina Paschinger
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biology ,Glycobiology ,biology.organism_classification ,Caenorhabditis elegans ,Cell biology - Published
- 2021
9. A New Era of Discovery in Carbohydrate Chemistry
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Jianglong Zhu, Xuechen Li, Pierre-Alexandre Driguez, Todd L. Lowary, and Nicola L. B. Pohl
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Biochemistry ,Carbohydrate chemistry ,Chemistry ,Organic Chemistry - Published
- 2020
10. A Very Short History of the Carbohydrate Division of the American Chemical Society
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Todd L. Lowary and Nicola L. B. Pohl
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Chemistry ,Organic Chemistry ,Division (horticulture) ,Carbohydrates ,Library science ,United States ,Chemical society - Published
- 2020
11. Structural insight into the binding of human galectins to corneal keratan sulfate, its desulfated form and related saccharides
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Nicola L. B. Pohl, Sayantan Bhaduri, Robert J. Linhardt, Kevin H. Mayo, Chao Cai, Hans-Joachim Gabius, Michelle C. Miller, Kanin Wichapong, Biochemie, and RS: Carim - B01 Blood proteins & engineering
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0301 basic medicine ,Keratan sulfate ,Galectins ,Disaccharide ,Biophysics ,lcsh:Medicine ,Plasma protein binding ,Molecular Dynamics Simulation ,Biochemistry ,Article ,Glycosaminoglycan ,03 medical and health sciences ,chemistry.chemical_compound ,Molecular recognition ,DIVERSE ROLES ,CHONDROITIN SULFATE ,Humans ,GLYCAN RECOGNITION ,CELL ,Binding site ,lcsh:Science ,Nuclear Magnetic Resonance, Biomolecular ,Galectin ,POLY-N-ACETYLLACTOSAMINE ,Glycosaminoglycans ,Multidisciplinary ,Binding Sites ,030102 biochemistry & molecular biology ,biology ,GLYCOSYLATION ,lcsh:R ,Lectin ,LECTIN ,030104 developmental biology ,chemistry ,P53-INDUCED GENE-1 ,Keratan Sulfate ,biology.protein ,lcsh:Q ,LIGANDS ,Proteoglycans ,Protein Binding - Abstract
Glycosaminoglycan chains of keratan sulfate proteoglycans appear to be physiologically significant by pairing with tissue lectins. Here, we used NMR spectroscopy and molecular dynamics (MD) simulations to characterize interactions of corneal keratan sulfate (KS), its desulfated form, as well as di-, tetra- (N-acetyllactosamine and lacto-N-tetraose) and octasaccharides with adhesion/growth-regulatory galectins, in particular galectin-3 (Gal-3). The KS contact region involves the lectin canonical binding site, with estimated KD values in the low µM range and stoichiometry of ~ 8 to ~ 20 galectin molecules binding per polysaccharide chain. Compared to Gal-3, the affinity to Gal-7 is relatively low, signaling preferences among galectins. The importance of the sulfate groups was delineated by using desulfated analogs that exhibit relatively reduced affinity. Binding studies with two related di- and tetrasaccharides revealed a similar decrease that underscores affinity enhancement by repetitive arrangement of disaccharide units. MD-based binding energies of KS oligosaccharide-loaded galectins support experimental data on Gal-3 and -7, and extend the scope of KS binding to Gal-1 and -9N. Overall, our results provide strong incentive to further probe the relevance of molecular recognition of KS by galectins in terms of physiological processes in situ, e.g. maintaining integrity of mucosal barriers, intermolecular (lattice-like) gluing within the extracellular meshwork or synaptogenesis.
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- 2020
12. Addition of Sialic Acid to Insulin Confers Superior Physical Properties and Bioequivalence
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Fa Liu, Daniel E. K. Kabotso, Nicola L. B. Pohl, David L. Smiley, Richard D. DiMarchi, Vasily M. Gelfanov, Diego Perez-Tilve, and John P. Mayer
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Drug ,Blood Glucose ,Male ,medicine.medical_treatment ,media_common.quotation_subject ,Peptide ,Diabetes Mellitus, Experimental ,chemistry.chemical_compound ,Mice ,Drug Discovery ,medicine ,Animals ,Humans ,Insulin ,Receptor ,media_common ,chemistry.chemical_classification ,HEK 293 cells ,N-Acetylneuraminic Acid ,Receptor, Insulin ,Sialic acid ,Mice, Inbred C57BL ,HEK293 Cells ,chemistry ,Biochemistry ,Therapeutic Equivalency ,Sialic Acids ,Molecular Medicine ,Oligopeptides ,Conjugate ,Hormone - Abstract
Native insulin is susceptible to biophysical aggregation and fibril formation, promoted by manual agitation and elevated temperatures. The safety of the drug and its application to alternative forms of administration could be enhanced through the identification of chemical modifications that strengthen its physical stability without compromising its biological properties. Complex polysialic acids (PSAs) exist naturally and provide a means to enhance the physical properties of peptide therapeutics. A set of insulin analogues site-specifically derivatized with sialic acid were prepared in an overall yield of 50-60%. Addition of a single or multiple sialic acids conferred remarkable enhancement to the biophysical stability of human insulin while maintaining its potency. The time to the onset of fibrillation was extended by more than 10-fold relative to that of the native hormone. These results demonstrate that simplified sialic acid conjugates represent a viable alternative to complex natural PSAs in increasing the stability of therapeutic peptides.
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- 2020
13. Modular Continuous Flow Synthesis of Orthogonally Protected 6-Deoxy Glucose Glycals
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Nicola L. B. Pohl, Clay S. Bennett, Subbarao Yalamanchili, and Tu-Anh V Nguyen
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Reaction conditions ,Time Factors ,business.industry ,Continuous flow ,Chemistry ,Organic Chemistry ,Oligosaccharides ,Chemistry Techniques, Synthetic ,Modular design ,Deoxyglucose ,Biochemistry ,Combinatorial chemistry ,Article ,Cold Temperature ,Kinetics ,Flow (mathematics) ,Yield (chemistry) ,Active cooling ,Physical and Theoretical Chemistry ,business ,Protecting group ,Flow time ,Ethers - Abstract
An efficient, modular continuous flow process towards accessing two orthogonally protected glycals is described with the development of reaction conditions for several common protecting group additions in flow, including the addition of benzyl, naphthylmethyl and tert-butyldimethylsilyl ethers. The process affords the desired target compounds in 57-74% overall yield in just 21-37 minutes of flow time. Furthermore, unlike batch conditions, the flow processes avoided the need for active cooling to prevent unwanted exotherms and required shorter reaction times.
- Published
- 2020
14. Parallel Glyco-SPOT Synthesis of Glycopeptide Libraries
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Michael Boyce, Christoffer K. Goth, Ravi Kumar H. Veeraiah, Akul Y. Mehta, Robert Kardish, Melinda S. Hanes, Nicola L. B. Pohl, Chao Gao, Richard D. Cummings, Yasuyuki Matsumoto, Sucharita Dutta, Kathrin Stavenhagen, and Jamie Heimburg-Molinaro
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Glycan ,Glycosylation ,Clinical Biochemistry ,Peptide ,Fluorescence Polarization ,01 natural sciences ,Biochemistry ,Antibodies ,Article ,chemistry.chemical_compound ,Peptide Library ,Polysaccharides ,Drug Discovery ,Molecular Biology ,Chromatography, High Pressure Liquid ,Pharmacology ,chemistry.chemical_classification ,biology ,010405 organic chemistry ,Glycopeptides ,Glycosyltransferases ,Microarray Analysis ,Receptor–ligand kinetics ,Glycopeptide ,0104 chemical sciences ,chemistry ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,biology.protein ,Molecular Medicine ,DNA microarray ,Glycoprotein ,Fluorescence anisotropy - Abstract
Glycan recognition is typically studied using free glycans, but glycopeptide presentations represent more physiological conditions for glycoproteins. To facilitate studies of glycopeptide recognition, we developed Glyco-SPOT synthesis, which enables the parallel production of diverse glycopeptide libraries at microgram scales. The method uses a closed system for prolonged reactions required for coupling Fmoc-protected glycoamino acids, including O-, N-, and S-linked glycosides, and release conditions to prevent side reactions. To optimize reaction conditions and sample reaction progress, we devised a biopsy testing method. We demonstrate the efficient utilization of such microscale glycopeptide libraries to determine the specificity of glycan-recognizing antibodies (e.g., CTD110.6) using microarrays, enzyme specificity on-array and in-solution (e.g., ST6GalNAc1, GCNT1, and T-synthase), and binding kinetics using fluorescence polarization. We demonstrated that the glycosylation on these peptides can be expanded using glycosyltransferases both in-solution and on-array. This technology will promote the discovery of biological functions of peptide modifications by glycans.
- Published
- 2020
15. Challenges in the Conversion of Manual Processes to Machine-Assisted Syntheses: Activation of Thioglycoside Donors with Aryl(trifluoroethyl)iodonium Triflimide
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Nicola L. B. Pohl, Gabriel A. Nieves, Regis C. Saliba, Zachary Wooke, An-Hsiang Adam Chu, and Clay S. Bennett
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Glycosylation ,Molecular Structure ,Cyclohexanones ,010405 organic chemistry ,Chemistry ,Aryl ,Organic Chemistry ,Glycosyl acceptor ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Combinatorial chemistry ,Article ,Scavenger (chemistry) ,0104 chemical sciences ,chemistry.chemical_compound ,Thioglycosides ,Orthoester ,Physical and Theoretical Chemistry ,Protecting group ,Oligosaccharide synthesis - Abstract
The steps needed to adapt a stable iodonium promoter for use in automated fluorous-assisted solution-phase oligosaccharide synthesis are described. Direct adaptation of the originally reported batch procedure resulted in the formation of an orthoester or protecting group transfer to the glycosyl acceptor. Fortunately, the addition of inexpensive β-pinene as an acid scavenger avoided both of these side reactions. The utility of this newly developed protocol was applied to the automated solution-phase synthesis of a β-glucan fragment.
- Published
- 2018
16. Robots command enzymes
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Nicola L. B. Pohl
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chemistry.chemical_classification ,Glycan ,biology ,010405 organic chemistry ,Chemistry ,General Chemical Engineering ,General Chemistry ,Computational biology ,010402 general chemistry ,01 natural sciences ,0104 chemical sciences ,Enzyme ,Homogeneous ,biology.protein - Abstract
Enzymatic approaches to synthesize oligosaccharides offer an alternative to chemical syntheses for the production of homogeneous glycans; however, enzyme-based routes typically require lengthy processes. Now, the design of a water-soluble affinity tag has enabled the automation of multistep enzymatic syntheses of mammalian oligosaccharides.
- Published
- 2019
17. Identification and deconvolution of carbohydrates with gas chromatography-vacuum ultraviolet spectroscopy
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Jonathan Smuts, Nicola L. B. Pohl, Kevin A. Schug, Jamie Schenk, Allegra Leghissa, and Gabe Nagy
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Analyte ,Anomer ,Vacuum ,Carbohydrates ,010402 general chemistry ,Mass spectrometry ,Methylation ,01 natural sciences ,Biochemistry ,Gas Chromatography-Mass Spectrometry ,Analytical Chemistry ,chemistry.chemical_compound ,Fragmentation (mass spectrometry) ,Oximes ,Derivatization ,chemistry.chemical_classification ,Chromatography ,010401 analytical chemistry ,Organic Chemistry ,General Medicine ,Silanes ,Furanose ,0104 chemical sciences ,Pharmaceutical Preparations ,chemistry ,Pyranose ,Spectrophotometry, Ultraviolet ,Gas chromatography - Abstract
Methodology for qualitative and quantitative determination of carbohydrates with gas chromatography coupled to vacuum ultraviolet detection (GC-VUV) is presented. Saccharides have been intently studied and are commonly analyzed by gas chromatography-mass spectrometry (GC-MS), but not always effectively. This can be attributed to their high degree of structural complexity: α/β anomers from their axial/equatorial hydroxyl group positioning at the C1-OH and flexible ring structures that lead to the open chain, five-membered ring furanose, and six-membered ring pyranose configurations. This complexity can result in convoluted chromatograms, ambiguous fragmentation patterns and, ultimately, analyte misidentification. In this study, mono-, di, and tri-saccharides were derivatized by two different methods-permethylation and oximation/pertrimethylsilylation-and analyzed by GC-VUV. These two derivatization methods were then compared for their efficiency, ease of use, and robustness. Permethylation proved to be a useful technique for the analysis of ketopentoses and pharmaceuticals soluble in dimethyl sulfoxide (DMSO), while the oximation/pertrimethylsilylation method prevailed as the more promising, overall, derivatization method. VUV spectra have been shown to be distinct and allow for efficient differentiation of isomeric species such as ketopentoses and reducing versus non-reducing sugars. In addition to identification, pharmaceutical samples containing several compounds were derivatized and analyzed for their sugar content with the GC-VUV technique to provide data for qualitative analysis.
- Published
- 2017
18. Pentavalent Bismuth as a Universal Promoter for S-Containing Glycosyl Donors with a Thiol Additive
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Daniel E. K. Kabotso and Nicola L. B. Pohl
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chemistry.chemical_classification ,Glycosylation ,010405 organic chemistry ,Chemistry ,Organic Chemistry ,Propanethiol ,chemistry.chemical_element ,Glycoside ,Uronic acid ,010402 general chemistry ,01 natural sciences ,Biochemistry ,0104 chemical sciences ,Bismuth ,Sialic acid ,carbohydrates (lipids) ,chemistry.chemical_compound ,Thiol ,Organic chemistry ,Glycosyl ,Physical and Theoretical Chemistry - Abstract
S-Propyl glycosides of less activated sugars, such as peracetylated carbohydrates and uronic acid esters that could not previously be activated with triphenylbismuth ditriflate alone, were found to be glycosylated in the presence of propanethiol as an additive in under 3 h. This newly developed protocol was also found to be effective in promoting glycosylation of neutral and uronic acid esters of S-phenyl, S-thiazolinyl, S-benzoxazolyl, and S-adamantyl glycosides as well as sialic acid.
- Published
- 2017
19. Functionalization promotes pathogen-mimicking characteristics of polyanhydride nanoparticle adjuvants
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Nicola L. B. Pohl, Balaji Narasimhan, Yashdeep Phanse, Brenda R. Carrillo-Conde, Michael J. Wannemuehler, Scott Broderick, Krishna Rajan, Rajarshi Roychoudhury, Bryan H. Bellaire, and Amanda E. Ramer-Tait
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0301 basic medicine ,Innate immune system ,Materials science ,biology ,medicine.medical_treatment ,Metals and Alloys ,Biomedical Engineering ,Rational design ,02 engineering and technology ,021001 nanoscience & nanotechnology ,Cell biology ,Biomaterials ,03 medical and health sciences ,030104 developmental biology ,Immune system ,Adjuvanticity ,Immunology ,MHC class I ,Ceramics and Composites ,medicine ,biology.protein ,0210 nano-technology ,Adjuvant ,Linker ,Intracellular - Abstract
Rational design of adjuvants and delivery systems will promote development of next-generation vaccines to control emerging and re-emerging diseases. To accomplish this, understanding the immune-enhancing properties of new adjuvants relative to those induced by natural infections can help with the development of pathogen-mimicking materials that will effectively initiate innate immune signaling cascades. In this work, the surfaces of polyanhydride nanoparticles composed of sebacic acid (SA) and 1,6-bis(p-carboxyphenoxy) hexane were decorated with an ethylene diamine spacer partially modified with either a glycolic acid linker or an α-1,2-linked di-mannopyranoside (di-mannose) to confer "pathogen-like" properties and enhance adjuvanticity. Co-incubation of linker-modified nanoparticles with dendritic cells (DCs) elicited significant increases in surface expression of MHC I, MHC II, CD86, and CD40, and enhanced secretion of IL-6, IL-12p40, and TNF-α. An 800% increase in uptake of ethylene-diamine-spaced, linker and di-mannose functionalized polyanhydride nanoparticles was also observed. Together, our data showed that linker-functionalized polyanhydride nanoparticles demonstrate similar patterns of uptake, intracellular trafficking, particle persistence, and innate activation as did DCs exposed to Yersinia pestis or Escherichia coli. These results set the stage for rational selection of adjuvant chemistries to induce pathogen-mimicking immune responses. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2762-2771, 2017.
- Published
- 2017
20. Recent Advances in the Analysis of Complex Glycoproteins
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Gabe Nagy, Stefan Gaunitz, Nicola L. B. Pohl, and Milos V. Novotny
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0301 basic medicine ,chemistry.chemical_classification ,Chemistry ,010401 analytical chemistry ,Analytic Sample Preparation Methods ,Computational biology ,01 natural sciences ,Chemistry Techniques, Analytical ,Article ,0104 chemical sciences ,Analytical Chemistry ,03 medical and health sciences ,030104 developmental biology ,Animals ,Humans ,Glycoprotein ,Glycoproteins - Published
- 2016
21. Designing sugar mimetics: non-natural pyranosides as innovative chemical tools
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Regis C. Saliba and Nicola L. B. Pohl
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chemistry.chemical_classification ,Magnetic Resonance Spectroscopy ,010405 organic chemistry ,Chemistry ,Biomolecule ,Molecular Mimicry ,Nanotechnology ,Ligands ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Article ,0104 chemical sciences ,Analytical Chemistry ,Carbohydrate Conformation ,Carbohydrate Metabolism ,Glycosides - Abstract
The importance of oligosaccharides in myriad biological processes is becoming increasingly clear. However, these carbohydrate-mediated processes are often challenging to dissect due to the often poor affinity, stability and selectivity of the oligosaccharides involved. To circumvent these issues, non-natural carbohydrates — carbohydrate mimics — are being designed as innovative tools to modify biomolecules of interest or to understand biological pathways using fluorescence microscopy, X-ray or nuclear magnetic resonance spectroscopy (NMR). This review focuses on key examples of recently developed non-natural sugars to answer specific biological needs.
- Published
- 2016
22. Automated solution-phase syntheses of alpha 1 → 2, 1 → 3 type rhamnans and rhamnan sulfate fragments
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Victoria R. Kohout and Nicola L. B. Pohl
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Trimethylsilyl ,Chemistry Techniques, Synthetic ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Article ,Analytical Chemistry ,Mannans ,chemistry.chemical_compound ,Automation ,Sulfation ,Deoxy Sugars ,Trisaccharide ,Sulfate ,chemistry.chemical_classification ,010405 organic chemistry ,Sulfates ,Organic Chemistry ,General Medicine ,Solution phase ,Combinatorial chemistry ,0104 chemical sciences ,Handling system ,Solutions ,chemistry ,Trifluoromethanesulfonate - Abstract
Rhamnan and rhamnan sulfate are naturally occurring carbohydrates that have important biological functions and possible therapeutic applications, but studies are limited to the microheterogeneous mixtures from natural sources. This work reports the first synthesis of any sulfated rhamnan fragments and successful automation of the process with a recently developed automated solution-phase approach using N-iodosuccinimide/trimethylsilyl triflate (NIS/TMSOTf) promotor and levulinoyl ester deprotection conditions. The automated solution-phase activation/deprotection approach was initially able to create alpha 1 → 2, 1 → 3 type rhamnan di- and trisaccharide in moderate yields. Once these targets were achieved, a process to use SO3•pyridine complex in DMF for sulfation compatible with an automated solution-phase liquid handling system was developed and successfully applied to carbohydrate sulfation to create two rhamnan sulfate fragments with differing monosulfation patterns.
- Published
- 2019
23. Probing deoxysugar conformational preference: A comprehensive computational study investigating the effects of deoxygenation
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Nicola L. B. Pohl and Alison E. Vickman
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Work (thermodynamics) ,Anomer ,Implicit solvation ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Article ,Analytical Chemistry ,symbols.namesake ,Polarizability ,Computational chemistry ,Oxidation state ,Deoxy Sugars ,Carbohydrate Conformation ,Deoxygenation ,Density Functional Theory ,010405 organic chemistry ,Chemistry ,Organic Chemistry ,General Medicine ,0104 chemical sciences ,Gibbs free energy ,Oxygen ,Pyranose ,symbols ,Thermodynamics - Abstract
Deoxysugars are intrinsic components in a number of antibiotics, antimicrobials, and therapeutic agents that often dictate receptor binding, improve efficacy, and provide a diverse toolbox in modifying glycoconjugate function due to an extensive number of unique isomers and inherent conformational flexibility. Hence, this work provides a comprehensive examination of the conformational effects associated with deoxygenation of the pyranose ring. Both the location and degree of deoxygenation were evaluated by interrogating the energetic landscape for a number of mono- and dideoxyhexopyranose derivatives using DFT methods (M05-2X/cc-pVTZ(-f)). Both anomeric forms and in some cases, the alternate chair form, have been investigated in the gas phase. As was documented in a preceding study, variation of the C-6 oxidation state has been shown to affect the anomeric preference of select glucose stereoisomers. Similar results were also observed for several deoxysugar isomers in this work, wherein the alternate anomer was favored upon reduction to the 6-deoxyhexose derivative or oxidation to the hexonic acid. Additionally, comparison of relative Gibbs free energies revealed C-3 deoxygenation imparts greater instability compared to C-2 or C-4 deoxygenation, as indicated by an increase in free energy for 3-deoxysugars. A polarizable continuum solvation model was also applied to empirically validate theoretical results for several deoxysugars, wherein good agreement with both carbon (σ = 1.6 ppm) and proton (σ = 0.20 ppm) NMR shifts was observed for the majority of isomers. Solvated and gas phase anomeric ratios were also calculated and compared favorably to reported literature values, although some discrepancies are noted.
- Published
- 2018
24. Multidimensional Analysis of 16 Glucose Isomers by Ion Mobility Spectrometry
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Gabe Nagy, Nicola L. B. Pohl, M. M. Gaye, and David E. Clemmer
- Subjects
chemistry.chemical_classification ,Ion-mobility spectrometry ,Spectrum Analysis ,Electrospray ionization ,010401 analytical chemistry ,Diastereomer ,Analytical chemistry ,Stereoisomerism ,010402 general chemistry ,Mass spectrometry ,01 natural sciences ,0104 chemical sciences ,Analytical Chemistry ,Ion ,Crystallography ,Glucose ,chemistry ,Carbohydrate Conformation ,Monosaccharide ,Enantiomer - Abstract
Diastereomeric adducts comprising an enantiomerically pure monosaccharide analyte, a peptide, and/or an amino acid and a divalent metal ion (for 16 different monosaccharide isomers) are generated by electrospray ionization and analyzed by combined ion mobility spectrometry-mass spectrometry (IMS-MS) techniques. Mobility distributions of [l-Ser + M + H](+) (where l-Ser is l-serine and M is a given monosaccharide), [l-Phe-Gly + M + H](+) (where l-Phe-Gly is l-phenylalanine-glycine), and [Mn(II) + (l-Phe-Gly - H) + M](+) complex ions are used to determine collision cross sections (ccs in Å(2)), and groups of cross sections for different clusters are proposed as means of identifying the sugar isomers. Within one type of complex, variations in ccs do not always allow delineation between the 16 glucose isomers, but interestingly, when ccs of three different ions are combined as a spatial vector, enantiomers are partially resolved. As a result of this analysis, l-glucose, d-glucose, l-allose, d-allose, d-gulose, d-galactose, and l-mannose are delineated, and for all eight enantiomeric pairs, d and l entities display different coordinates. In addition, different combinations of amino acids, peptide, and metal ions are surveyed, and the potential for yielding unique coordinates for the generated diastereomeric complexes is assessed.
- Published
- 2016
25. Introducing Students to Protein Analysis Techniques: Separation and Comparative Analysis of Gluten Proteins in Various Wheat Strains
- Author
-
Alyssa L. Pirinelli, Jonathan C. Trinidad, and Nicola L. B. Pohl
- Subjects
0301 basic medicine ,chemistry.chemical_classification ,Science instruction ,Computer science ,education ,05 social sciences ,Wheat flour ,food and beverages ,nutritional and metabolic diseases ,050301 education ,Context (language use) ,General Chemistry ,Gluten Proteins ,Data science ,Gluten ,Original research ,Education ,03 medical and health sciences ,030104 developmental biology ,Chemical engineering ,chemistry ,0503 education - Abstract
Polyacrylamide gel electrophoresis (PAGE) is commonly taught in undergraduate laboratory classes as a traditional method to analyze proteins. An experiment has been developed to teach these basic protein gel skills in the context of gluten protein isolation from various types of wheat flour. A further goal is to relate this technique to current mass spectrometry-based proteomic analysis techniques. Gluten proteins serve as particularly good experimental systems for the undergraduate classroom given the low cost of their sources and the current widespread public interest in gluten-free diets and Celiac-Sprue disease. The experiment set also can serve as preliminary data for student-written original research proposals and be the basis for a discussion of how scientific topics are presented in public forums. This experiment can also be readily adapted to a range of audiences and facilities.
- Published
- 2015
26. Automated Solution-Phase Synthesis of Insect Glycans to Probe the Binding Affinity of Pea Enation Mosaic Virus
- Author
-
Nicola L. B. Pohl, Lucas B. Linz, Shu-Lun Tang, and Bryony C. Bonning
- Subjects
Glycan ,Virus ,Article ,Mosaic Viruses ,Polysaccharides ,Animals ,RNA Viruses ,Amino Acid Sequence ,Viral Structural Proteins ,Mosaic virus ,biology ,Base Sequence ,Chemistry ,Organic Chemistry ,Peas ,RNA ,food and beverages ,Isothermal titration calorimetry ,RNA virus ,Pea enation mosaic virus ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,A-site ,Biochemistry ,biology.protein ,RNA, Viral - Abstract
Pea enation mosaic virus (PEMV)--a plant RNA virus transmitted exclusively by aphids--causes disease in multiple food crops. However, the aphid-virus interactions required for disease transmission are poorly understood. For virus transmission, PEMV binds to a heavily glycosylated receptor aminopeptidase N in the pea aphid gut and is transcytosed across the gut epithelium into the aphid body cavity prior to release in saliva as the aphid feeds. To investigate the role of glycans in PEMV-aphid interactions and explore the possibility of viral control through blocking a glycan interaction, we synthesized insect N-glycan terminal trimannosides by automated solution-phase synthesis. The route features a mannose building block with C-5 ester enforcing a β-linkage, which also provides a site for subsequent chain extension. The resulting insect N-glycan terminal trimannosides with fluorous tags were used in a fluorous microarray to analyze binding with fluorescein isothiocyanate-labeled PEMV; however, no specific binding between the insect glycan and PEMV was detected. To confirm these microarray results, we removed the fluorous tag from the trimannosides for isothermal titration calorimetry studies with unlabeled PEMV. The ITC studies confirmed the microarray results and suggested that this particular glycan-PEMV interaction is not involved in virus uptake and transport through the aphid.
- Published
- 2015
27. Etymology of the elements Antimony, Gold, and Jupiter's Wolf: How the Elements Were Named Peter Wothers Oxford University Press, 2020. 304 pp
- Author
-
Nicola L. B. Pohl
- Subjects
Literature ,Multidisciplinary ,business.industry ,Etymology ,Chemistry (relationship) ,Element (category theory) ,business ,Simple (philosophy) - Abstract
Peter Wothers, a teaching fellow at the University of Cambridge, is interested in the names of elements whose origins are murky. And while a simple web search can provide the etymologies of element names, his new book, Antimony, Gold, and Jupiter9s Wolf provides tales about how those names came to be.
- Published
- 2020
28. Putting sugars under strain
- Author
-
Nicola L. B. Pohl
- Subjects
Multidisciplinary ,Strain (chemistry) ,Chemistry ,Food science ,Article - Abstract
Cyclodextrins (CDs) are cyclic oligomers of α-1,4-d-glucopyranoside and are known mainly as hexamers to octamers. The central cavities of CDs can retain small molecules, enabling diverse applications. The smallest members, CD3 and CD4, have ring sizes too small to permit the most stable conformations of glucopyranose and have not been accessible synthetically. In this study, we present methods to chemically synthesize both CD3 and CD4. The main factor in the successful synthesis is the creation of a glucopyranose ring conformationally counterbalanced between equatorial- and axial-rich forms. This suppleness is imparted by a bridge between O-3 and O-6 of glucose, which enables the generation of desirable, albeit deformed, conformers when synthesizing the cyclic trimer and tetramer.
- Published
- 2019
29. Leishmania-Derived Trimannose Modulates the Inflammatory Response To Significantly Reduce Leishmania major-Induced Lesions
- Author
-
Molly Parrish, Chelsea L. Rintelmann, Katherine N. Gibson-Corley, Angela J. Toepp, Tara Grinnage-Pulley, Robert G. Schaut, Daniel E. K. Kabotso, Nicola L. B. Pohl, Rajarshi Roychoudhury, and Christine A. Petersen
- Subjects
0301 basic medicine ,Male ,030231 tropical medicine ,Immunology ,Leishmaniasis, Cutaneous ,Receptors, Cell Surface ,Interferon-gamma secretion ,Biology ,Microbiology ,Proinflammatory cytokine ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,Cutaneous leishmaniasis ,medicine ,Animals ,Leishmania major ,Lectins, C-Type ,Latex beads ,Mice, Knockout ,Lipophosphoglycan ,medicine.disease ,biology.organism_classification ,Leishmania ,Microspheres ,Mice, Inbred C57BL ,030104 developmental biology ,Infectious Diseases ,Mannose-Binding Lectins ,chemistry ,Parasitology ,Female ,Fungal and Parasitic Infections ,Mannose ,Mannose receptor ,Mannose Receptor - Abstract
Leishmania lipophosphoglycan (LPG) is a key virulence factor, initiating inflammation resulting in cutaneous lesions. LPG is capped by various oligosaccharides. How these glycans are recognized and how they alter the course of Leishmania infection are poorly understood. Previous studies synthesized α-1,2-trimannose cap sugars on latex beads and demonstrated that C57BL/6 mice coinoculated with Leishmania major and trimannose-coated beads produced significantly higher levels of interleukin-12p40 (IL-12p40) and other proinflammatory, type 1 cytokines than mice inoculated with L. major alone within the first 48 h of infection. However, as L. major infection typically progress over weeks to months, the role of trimannose in altering disease progression over the course of infection was unknown. Wild-type mice were inoculated with either trimannose-coated or carrier (uncoated) beads, infected with L. major alone, coinoculated with carrier beads and L. major , or coinoculated with trimannose-coated beads and L. major . Trimannose treatment of L. major -infected mice decreased the parasite load and significantly decreased the lesion size at 14 days postinfection (p.i.) compared to results for nontreated, infected mice. Infected, trimannose-treated mice had decreased IL-12p40 and IL-10 secretion and increased interferon gamma secretion at 14 days p.i. Mannose receptor knockout (MR −/− ) mice lack the ability to detect trimannose. When MR −/− mice were infected with L. major and treated with trimannose beads, they did not have decreased lesion size. Leishmania -derived trimannose represents a novel immunomodulator that provides early type 1-skewed cytokine production to control the parasite load and alter the course of cutaneous leishmaniasis.
- Published
- 2017
30. Scope and limitations of carbohydrate hydrolysis for de novo glycan sequencing using a hydrogen peroxide/metallopeptide-based glycosidase mimetic
- Author
-
Tianyuan Peng, Zachary Wooke, and Nicola L. B. Pohl
- Subjects
Glycan ,Analyte ,Glycoside Hydrolases ,Carbohydrates ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Article ,Analytical Chemistry ,chemistry.chemical_compound ,Hydrolysis ,Polysaccharides ,Monosaccharide ,Glycoside hydrolase ,Hydrogen peroxide ,chemistry.chemical_classification ,biology ,010405 organic chemistry ,Organic Chemistry ,General Medicine ,Hydrogen Peroxide ,Oligosaccharide ,Combinatorial chemistry ,0104 chemical sciences ,chemistry ,biology.protein ,Acid hydrolysis - Abstract
Acidic hydrolysis is commonly used as a first step to break down oligo- and polysaccharides into monosaccharide units for structural analysis. While easy to set up and amenable to mass spectrometry detection, acid hydrolysis is not without its drawbacks. For example, ring-destruction side reactions and degradation products, along with difficulties in optimizing conditions from analyte to analyte, greatly limits its broad utility. Herein we report studies on a hydrogen peroxide/CuGGH metallopeptide-based glycosidase mimetic design for a more efficient and controllable carbohydrate hydrolysis. A library of methyl glycosides consisting of ten common monosaccharide substrates, along with oligosaccharide substrates, was screened with the artificial glycosidase for hydrolytic activity in a high-throughput format with a robotic liquid handling system. The artificial glycosidase was found to be active towards most screened linkages, including alpha- and beta-anomers, thus serving as a potential alternative method for traditional acidic hydrolysis approaches of oligosaccharides.
- Published
- 2017
31. Introduction: Carbohydrate Chemistry
- Author
-
Nicola L. B. Pohl
- Subjects
Chemical Phenomena ,010405 organic chemistry ,Chemistry ,Carbohydrate chemistry ,Extramural ,Carbohydrates ,Chemistry, Organic ,Library science ,General Chemistry ,010402 general chemistry ,01 natural sciences ,Article ,0104 chemical sciences ,Introductory Journal Article - Published
- 2018
32. Acid-Triggered Degradable Reagents for Differentiation of Adaptive and Innate Immune Responses toLeishmania-Associated Sugars
- Author
-
Rajarshi Roychoudhury, Robert G. Schaut, Tara Grinnage-Pulley, Nicola L. B. Pohl, Christine A. Petersen, and Pedro A. Martinez
- Subjects
Lipopolysaccharides ,T-Lymphocytes ,Adaptive Immunity ,Catalysis ,Antigen ,Humans ,Macrophage ,Receptor ,Leishmaniasis ,Pathogen ,Leishmania ,Latex beads ,Antigen Presentation ,Innate immune system ,biology ,Chemistry ,Macrophages ,Biological activity ,General Medicine ,General Chemistry ,biology.organism_classification ,Immunity, Innate ,Biochemistry ,Immunologic Techniques ,Indicators and Reagents ,Acids - Abstract
Lipopolysaccharides (LPS) of Leishmania spp are known to alter innate immune responses. However, the ability of these sugars to specifically alter adaptive T-cell responses is unclear. To study cap sugar-T-cell interactions, pathogen mimics (namely glycodendrimer-coated latex beads with acid-labile linkers) were synthesized. Upon lysosomal acidification, linker breakdown releases glycodendrimers for possible loading on antigen presenting molecules to induce T-cell growth. T-cell proliferation was indeed higher after macrophage exposure to mannobioside or -trioside-containing glycodendrimers than to non-functionalized beads. Yet, blocking phagolysosomal acidification only reduced T-cell proliferation with macrophages exposed to beads with an acid-labile-linker and not to covalently-linked beads. These sugar-modified reagents show that oligosaccharides alone can drive T-cell proliferation by acidification-requiring presentation, most significantly in NKT receptor (CD160)-restricted T cells.
- Published
- 2015
33. Automated Solution-Phase Synthesis of β-1,4-Mannuronate and β-1,4-Mannan
- Author
-
Shu-Lun Tang and Nicola L. B. Pohl
- Subjects
Letter ,Glycosylation ,Molecular Structure ,A-hexasaccharide ,Chemistry ,Stereochemistry ,Hexuronic Acids ,Organic Chemistry ,Biochemistry ,Combinatorial chemistry ,Mannans ,Solutions ,Automation ,chemistry.chemical_compound ,Homogeneous ,Molecule ,Carboxylate ,Physical and Theoretical Chemistry ,Glycosyl donor ,Solution phase synthesis ,Mannan - Abstract
The first automated solution-phase synthesis of β-1,4-mannuronate and β-1,4-mannan oligomers has been accomplished by using a β-directing C-5 carboxylate strategy. By utilizing fluorous-tag assisting purification after repeated reaction cycles, β-1,4-mannuronate was synthesized up to a hexasaccharide with limited loading of a glycosyl donor (up to 3.5 equiv) for each glycosylation cycle due to the homogeneous solution-phase reaction condition. After a global reduction of the uronates, the β-1,4-mannan hexasaccharide was obtained, thereby demonstrating a new approach to β-mannan synthesis.
- Published
- 2015
34. Monosaccharide Identification as a First Step toward de Novo Carbohydrate Sequencing: Mass Spectrometry Strategy for the Identification and Differentiation of Diastereomeric and Enantiomeric Pentose Isomers
- Author
-
Nicola L. B. Pohl and Gabe Nagy
- Subjects
chemistry.chemical_classification ,Xylulose ,Lyxose ,Ribulose ,Pentoses ,Pentose ,Stereoisomerism ,Furanose ,Mass Spectrometry ,Analytical Chemistry ,chemistry.chemical_compound ,Carbohydrate Sequence ,chemistry ,Ribose ,Monosaccharide ,Organic chemistry - Abstract
De novo carbohydrate sequencing, including monosaccharide identification, largely remains a tremendous analytical challenge. A first step in the complete structural determination of any large polysaccharide is an accurate and robust method for analysis of the constituent monosaccharides. Herein, the first mass spectrometry-based method for the complete identification and absolute configuration determination of all 12 pentose isomers, including the d and l enantiomers for arabinose, lyxose, ribose, xylose, ribulose, and xylulose, is reported. As compared to earlier work to distinguish hexose isomers, the chiral separation of the pentose isomers was significantly more challenging. Specifically, the 12 pentoses are much more structurally similar to one another, with only the axial or equatorial orientation of two hydroxyl groups differentiating among these isomers in their five-membered ring furanose structure and smaller energetic differences between pentose conformations than between hexose conformations. Despite such inherently minimal energetic differences between the 12 pentoses, two unique fixed ligand kinetic method combinations were discovered to achieve chiral discrimination for this set of isomers. This assay can be readily applied to the identification of any isolated pentose monosaccharide using only microgram quantities and a commercial instrument and complements the method to distinguish hexose isomers. A workflow that incorporates this mass spectrometry-based method and thereby could achieve complete de novo identification of all monosaccharide building blocks in an oligo- or polysaccharide is proposed.
- Published
- 2015
35. Safety and Biocompatibility of Carbohydrate-Functionalized Polyanhydride Nanoparticles
- Author
-
Rajarshi Roychoudhury, Paola M. Boggiatto, Nicola L. B. Pohl, Julia E. Vela-Ramirez, Michael J. Wannemuehler, Balaji Narasimhan, Jesse M. Hostetter, and Jonathan T. Goodman
- Subjects
Chemokine ,medicine.medical_treatment ,Carbohydrates ,Pharmaceutical Science ,Kidney ,Injections ,Polyethylene Glycols ,Mice ,Immune system ,In vivo ,Polyanhydrides ,medicine ,Animals ,Hexanes ,Lectins, C-Type ,Lung ,Administration, Intranasal ,Innate immune system ,biology ,Chemistry ,Pattern recognition receptor ,Galactose ,medicine.disease ,Glycolates ,Cellular infiltration ,Cytokine ,Liver ,Biochemistry ,biology.protein ,Cytokines ,Nanoparticles ,Female ,Nasal administration ,Chemokines ,Bronchoalveolar Lavage Fluid ,Mannose ,Research Article - Abstract
Carbohydrate functionalization of nanoparticles allows for targeting of C-type lectin receptors. This family of pattern recognition receptors expressed on innate immune cells, such as macrophages and dendritic cells, can be used to modulate immune responses. In this work, the in vivo safety profile of carbohydrate-functionalized polyanhydride nanoparticles was analyzed following parenteral and intranasal administration in mice. Polyanhydride nanoparticles based on 1,6-bis-(p-carboxyphenoxy)hexane and 1,8-bis-(p-carboxyphenoxy)-3,6-dioxaoctane were used. Nanoparticle functionalization with di-mannose (specifically carboxymethyl-α-D-mannopyranosyl-(1,2)-D-mannopyranoside), galactose (specifically carboxymethyl-β-galactoside), or glycolic acid induced no adverse effects after administration based on histopathological evaluation of liver, kidneys, and lungs. Regardless of the polymer formulation, there was no evidence of hepatic or renal damage or dysfunction observed in serum or urine samples. The histological profile of cellular infiltration and the cellular distribution and kinetics in the lungs of mice administered with nanoparticle treatments followed similar behavior as that observed in the lungs of animals administered with saline. Cytokine and chemokine profiles in bronchoalveolar lavage fluid indicated surface chemistry dependence on modest secretion of IL-6, IP-10, and MCP-1; however, there was no evidence of any deleterious histopathological changes. Based on these analyses, carbohydrate-functionalized nanoparticles are safe for in vivo applications. These results provide foundational information towards the evaluation of the capabilities of these surface-modified nanoparticles as vaccine delivery formulations.
- Published
- 2014
36. Nanoparticle Chemistry and Functionalization Differentially Regulates Dendritic Cell-Nanoparticle Interactions and Triggers Dendritic Cell Maturation
- Author
-
Michael J. Wannemuehler, Jonathan T. Goodman, Nicola L. B. Pohl, Balaji Narasimhan, Rajarshi Roychoudhury, Julia E. Vela Ramirez, and Paola M. Boggiatto
- Subjects
Chemistry ,Nanoparticle ,General Chemistry ,Dendritic cell ,Condensed Matter Physics ,Adsorption ,Biophysics ,Organic chemistry ,Surface modification ,General Materials Science ,Cytokine secretion ,Antigen-presenting cell ,Polyanhydrides ,Protein adsorption - Abstract
Biodegradable polyanhydride nanoparticles have been used as vaccine adjuvants and delivery vehicles. Upon parenteral administration, the surface characteristics of these nanoparticles may be modified by serum protein adsorption, likely affecting how the particles interact with antigen presenting cells. These studies investigated the role of the differential adsorption of serum proteins onto carbohydrate-functionalized and non-functionalized polyanhydride nanoparticles of different chemistries on interactions with dendritic cells (DCs). Di-mannose (a carboxymethyl-modified disaccharide of 1,2-α-linked-d-mannopyranoside) or glycolic acid was covalently linked to nanoparticles synthesized using copolymers of 1,6-bis(p-carboxyphenoxy)hexane (CPH) and sebacic acid (SA) and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) and CPH. Serum protein adsorption to CPH:SA nanoparticles enhanced particle internalization, but did not enhance uptake of CPTEG:CPH nanoparticles. When DCs were cultured with nanoparticles, the surface expression of MHC II, CD86, and CD206 was enhanced and mediated by both polymer chemistry and functionalization. Cytokine secretion by DCs was differentially modulated by both polymer chemistry and functionalization. The amount and profile of serum proteins adsorbed indicated that polymer hydrophobicity and functionalization enhanced protein adsorption. Overall polymer chemistry, functionalization, and serum protein adsorption influenced nanoparticle interactions with DCs and elucidating the complex relationships between these parameters and dendritic cell maturation will enable rational design of targeted vaccines and immunotherapies.
- Published
- 2014
37. Effects of Varying the 6-Position Oxidation State of Hexopyranoses: A Systematic Comparative Computational Analysis of 48 Monosaccharide Stereoisomers
- Author
-
Daniel C. Ashley, Alison E. Vickman, Mu-Hyun Baik, and Nicola L. B. Pohl
- Subjects
Steric effects ,Anomer ,010405 organic chemistry ,Chemistry ,Stereochemistry ,Organic Chemistry ,Cyclohexane conformation ,Substituent ,Carbohydrate ,010402 general chemistry ,01 natural sciences ,Catalysis ,Article ,0104 chemical sciences ,Computer Science Applications ,Inorganic Chemistry ,chemistry.chemical_compound ,Computational Theory and Mathematics ,Pyranose ,Oxidation state ,Electronic effect ,Physical and Theoretical Chemistry - Abstract
Knowledge of multi-dimensional carbohydrate structure is essential when delineating structure-function relationships in the development of analytical techniques such as ion mobility-mass spectrometry and of carbohydrate-based therapeutics, as well as in rationally modifying the chemical and physical properties of drugs and materials based on sugars. Although monosaccharides are conventionally presumed to adopt the canonical 4C1 chair conformation, it is not well known how altering the substituent identity around the pyranose ring affects the favored conformational state. This work provides a comprehensive and systematic computational comparison of all eight aldohexose isomers in the gas phase with reduction and oxidation at the C-6 position using density functional theory (M05-2X/cc-pVTZ(-f)//B3LYP/6-31G**) to determine the conformational and anomeric preference for each sugar in the gas phase. All 6-deoxyhexose and aldohexose isomers favored the 4C1 chair conformation, while oxidation at C-6 showed a shift in equilibrium to favor the 1C4 chair for β-alluronic acid, β-guluronic acid, and β-iduronic acid. The anomeric preference was found to be significantly affected by a remote change in oxidation state, with the alternate anomer favored for several isomers. These findings provide a fundamental platform to empirically test steric and electronic effects of pyranose substituents, with the goal of formulating straightforward rules that govern carbohydrate reactivity and drive quicker, more efficient syntheses. Graphical abstract A systematic comparative conformational analysis of all eight aldohexose isomers using DFT methods (M05-2X/cc-pVTZ(-f)) reveals changes in anomeric and ring conformational preference upon reduction or oxidation at the C-6 position for several sugars.
- Published
- 2017
38. Recent Liquid Chromatographic Approaches and Developments for the Separation and Purification of Carbohydrates
- Author
-
Tianyuan Peng, Nicola L. B. Pohl, and Gabe Nagy
- Subjects
Chromatography ,Chemistry ,General Chemical Engineering ,010401 analytical chemistry ,General Engineering ,Enzymatic synthesis ,010402 general chemistry ,01 natural sciences ,Article ,0104 chemical sciences ,Analytical Chemistry - Abstract
Carbohydrate purification remains a bottleneck in securing analytical standards from natural sources or by chemical or enzymatic synthesis. This review highlights the scope and remaining limitations of recent approaches and methods development in liquid chromatography for robust and higher-throughput carbohydrate separation and isolation.
- Published
- 2017
39. Regioselective Benzylation of 2-Deoxy-2-aminosugars using Crown Ethers: Application to a Shortened Synthesis of Hyaluronic Acid Oligomers
- Author
-
Lin Liu, Chinmoy Mukherjee, and Nicola L. B. Pohl
- Subjects
biology ,Regioselectivity ,General Chemistry ,biology.organism_classification ,Article ,chemistry.chemical_compound ,Benzyl bromide ,chemistry ,Glucosamine ,Sodium hydroxide ,Galactosamine ,Hyaluronic acid ,Tetra ,Organic chemistry ,Tetrahydrofuran - Abstract
The combination of benzyl bromide, sodium hydroxide and 15-crown-5 in tetrahydrofuran is shown to be an efficient method for installing benzyl groups at both the 4- and 6-positions regioselectively directly from peracetylated N-trichloroacetyl-protected glucosamine and galactosamine. Application of this benzylation strategy proved to significantly shorten the synthetic route to hyaluronic acid tetra- and hexamers.
- Published
- 2014
40. Synthesis of Fluorous Photolabile Aldehyde and Carbamate and Alkyl Carbamate Protecting Groups for Carbohydrate-Associated Amines
- Author
-
Rajarshi Roychoudhury and Nicola L. B. Pohl
- Subjects
chemistry.chemical_classification ,Aldehydes ,Carbamate ,Letter ,Molecular Structure ,medicine.medical_treatment ,Solid Phase Extraction ,Organic Chemistry ,Glycosyl acceptor ,Oligosaccharides ,Carbohydrate ,Biochemistry ,Aldehyde ,chemistry ,medicine ,Organic chemistry ,Molecule ,Indicators and Reagents ,Carbamates ,Solid phase extraction ,Amines ,Physical and Theoretical Chemistry ,Protecting group ,Alkyl - Abstract
Two new fluorous photolabile-protecting groups (FNBC and FNB) and a new base-labile protecting group (FOC) for the masking of amines are reported. The protecting groups survive a wide range of common reaction conditions used in oligosaccharide synthesis and render the attached molecules amenable to fluorous solid-phase extraction (FSPE). A glycosyl acceptor containing the FNB group is shown to be useful in the synthesis of carbohydrates tagged with free deactivated secondary amines.
- Published
- 2014
41. Protocol for the purification of protected carbohydrates: toward coupling automated synthesis to alternate-pump recycling high-performance liquid chromatography
- Author
-
Daniel E. K. Kabotso, Nicola L. B. Pohl, Gabe Nagy, Tianyuan Peng, and Milos V. Novotny
- Subjects
Chromatography ,010405 organic chemistry ,Chemistry ,fungi ,Metals and Alloys ,food and beverages ,General Chemistry ,010402 general chemistry ,01 natural sciences ,High-performance liquid chromatography ,Catalysis ,0104 chemical sciences ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,Materials Chemistry ,Ceramics and Composites ,Oligosaccharide synthesis - Abstract
Analytical techniques that can be coupled to automated oligosaccharide synthesis platforms are needed to purify to homogeneity protected carbohydrates at levels of ≥99.5% purity., Given recent advances in automated oligosaccharide synthesis, analytical techniques that can be coupled to a synthetic framework are needed to not just identify but also purify to homogeneity protected carbohydrate compounds at levels of ≥99.5% purity. Herein, an alternate-pump recycling high-performance liquid chromatography (R-HPLC) method has been developed to allow purification of protected carbohydrates at levels of ≥99.5% purity.
- Published
- 2016
42. The grand story of carbon Symphony in C: Carbon and the Evolution of (Almost) Everything Robert M. Hazen Norton, 2019. 313 pp
- Author
-
Nicola L. B. Pohl
- Subjects
Multidisciplinary ,chemistry ,Earth science ,chemistry.chemical_element ,Boundary (topology) ,Carbon - Abstract
Although organic chemistry is often described as the sci-ence of carbon, Robert Hazen9s latest book, Sym¬phony in C, makes clear that this vital element cannot be contained by such a disciplinary boundary.
- Published
- 2019
43. A mass-differentiated library strategy for identification of sugar nucleotidyltransferase activities from cell lysates
- Author
-
Lin Liu, Rahman M. Mizanur, Joy M. Jackson, Nicola L. B. Pohl, and Kwang-Seuk Ko
- Subjects
chemistry.chemical_classification ,Spectrometry, Mass, Electrospray Ionization ,Molecular Structure ,Biophysics ,Virulence ,Cell Biology ,Carbohydrate ,Biology ,Nucleotidyltransferase ,medicine.disease_cause ,Nucleotide sugar ,Nucleotidyltransferases ,Biochemistry ,Small Molecule Libraries ,chemistry.chemical_compound ,Enzyme ,chemistry ,Escherichia coli ,medicine ,Carbohydrate Metabolism ,Sugar ,Molecular Biology ,Pathogen - Abstract
Sugar nucleotidyltransferases, or nucleotide sugar pyrophosphorylases, are ubiquitous enzymes whose activities have been correlated to disease states and pathogen virulence. Here we report a rapid "one-pot" method to identify a range of sugar nucleotidyltransferase activities of purified proteins or in cell lysates using a mass-differentiated carbohydrate library designed for mass spectrometry-based analysis.
- Published
- 2013
44. General Label-Free Mass Spectrometry-Based Assay To Identify Glycosidase Substrate Competence
- Author
-
Gabe Nagy, Nicola L. B. Pohl, and Tianyuan Peng
- Subjects
0301 basic medicine ,Mass spectrometry ,Ligands ,01 natural sciences ,Mass Spectrometry ,Analytical Chemistry ,Substrate Specificity ,Mannans ,03 medical and health sciences ,Hydrolysis ,Mannobiose ,Escherichia coli ,Glycoside hydrolase ,Enzyme Assays ,chemistry.chemical_classification ,Beta-mannosidase ,biology ,Chemistry ,Thermus thermophilus ,010401 analytical chemistry ,Leaving group ,Psychosine ,beta-Mannosidase ,Stereoisomerism ,biology.organism_classification ,0104 chemical sciences ,Pyrococcus furiosus ,Canavalia ,Kinetics ,030104 developmental biology ,Enzyme ,Biochemistry ,alpha-Galactosidase - Abstract
Common glycosidase assays rely on the hydrolysis of non-natural labeled sugar substrates that thereby preclude obtaining information as to the specificity of the leaving group and therefore the most kinetically competent natural substrates. A β-mannosidase could be known to hydrolyze β-mannose, for example, but from what is presently hard to determine by any high-throughput means. Herein, the first chiral dopant-based mass spectrometric assay, with its foundation rooted in the Cooks’ fixed ligand kinetic method, is presented to screen label-free monosaccharide-containing substrates for their kinetic competency with a given glycosidase as a step to name these enzymes not just for the sugar that is removed but also for the leaving group that is produced. This work also presents the first information about the substrate specificity of two specific hyperthermophilic enzymes and the first test of some native, unlabeled substrates (α-1-4 mannobiose and β-1-galactosylphingosine) with mesophilic enzymes.
- Published
- 2016
45. Mechanistic Studies of Bismuth(V)-Mediated Thioglycoside Activation Reveal Differential Reactivity of Anomers
- Author
-
Mu-Hyun Baik, Nicola L. B. Pohl, Manibarsha Goswami, and Daniel C. Ashley
- Subjects
Glycosylation ,Anomer ,010405 organic chemistry ,Organic Chemistry ,chemistry.chemical_element ,010402 general chemistry ,01 natural sciences ,0104 chemical sciences ,Bismuth ,Chemical kinetics ,chemistry.chemical_compound ,chemistry ,Computational chemistry ,Reaction model ,Organic chemistry ,Reactivity (chemistry) ,Density functional theory ,Trifluoromethanesulfonate - Abstract
The mechanism of bismuth(V)-mediated thioglycoside activation was examined using reaction kinetics and quantum chemical reaction models. NMR experiments show an unusual nonlinear growth/decay curve for the glycosylation reaction. Further studies suggest an anomeric inversion of the β-glycoside donor to the α-donor during its activation, even in the presence of a neighboring 2-position acetate. Interestingly, in situ anomerization was not observed in the activation of an α-glycoside donor, and this anomer also showed faster reaction times and higher product diastereoselectivites. Density functional theory calculations identify the structure of the promoter triphenyl bismuth ditriflate, [Ph3Bi(OTf)2, 1], in solution and map out the energetics of its interactions with the two thioglycoside anomers. These calculations suggest that 1 must bind the thiopropyl arm to induce triflate loss. The computational analyses also show that, unlike most O-glycosides, the β- and α-donor S-glycosides are similar in energy. One energetically reasonable anomerization pathway of the donors is an SN1-like mechanism promoted by forming a bismuth-sulfonium adduct with the Lewis acidic Bi(V) for the formation of an oxacarbenium intermediate. Finally, the computed energy compensations needed to form these α vs β Bi adducts is a possible explanation for the differential reactivity of these donors.
- Published
- 2016
46. Triphenylbis(1,1,1-trifluoromethanesulfonato)-bismuth
- Author
-
Daniel E. K. Kabotso and Nicola L. B. Pohl
- Subjects
Chemistry ,chemistry.chemical_element ,Bismuth ,Nuclear chemistry - Published
- 2016
47. Fluorous-Tag Assisted Syntheses of Sulfated Keratan Sulfate Oligosaccharide Fragments
- Author
-
Nicola L. B. Pohl and Sayantan Bhaduri
- Subjects
Keratan sulfate ,Oligosaccharides ,Sulfuric Acid Esters ,010402 general chemistry ,01 natural sciences ,Biochemistry ,chemistry.chemical_compound ,Sulfation ,Organic chemistry ,Solid phase extraction ,Physical and Theoretical Chemistry ,Solubility ,chemistry.chemical_classification ,Fluorocarbons ,Molecular Structure ,010405 organic chemistry ,Organic Chemistry ,Extraction (chemistry) ,Solid Phase Extraction ,Oligosaccharide ,Combinatorial chemistry ,0104 chemical sciences ,carbohydrates (lipids) ,chemistry ,Keratan Sulfate - Abstract
The first block iteration strategy for iterative solution-phase synthesis of protected keratan sulfate (KS)-like fragments is reported. Obstacles in a strategy using galactose-glucosamine (Gal-GlcNAc) modules led to the discovery of a differentially protected GlcNAc-Gal module that could be used to synthesize KS-like fragments using a fluorous tag that maintained solubility in organic solvents for purification of all intermediates via fluorous solid-phase extraction.
- Published
- 2016
48. Automated fluorous-assisted solution-phase synthesis of β-1,2-, 1,3-, and 1,6-mannan oligomers
- Author
-
Shu-Lun Tang and Nicola L. B. Pohl
- Subjects
Glycosylation ,Carboxylic Acids ,Oligosaccharides ,Chemistry Techniques, Synthetic ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Article ,Analytical Chemistry ,Mannans ,chemistry.chemical_compound ,Automation ,Organic chemistry ,Glycosyl ,Hydroxymethyl ,Carboxylate ,Protecting group ,Mannan ,chemistry.chemical_classification ,010405 organic chemistry ,Chemistry ,Organic Chemistry ,Glycoside ,General Medicine ,Fluorine ,Combinatorial chemistry ,0104 chemical sciences ,carbohydrates (lipids) ,Solutions ,Solution phase synthesis - Abstract
Automated solution-phase syntheses of β-1,2-, 1,3-, and 1,6-mannan oligomers have been accomplished by applying a β-directing C-5 carboxylate strategy. Fluorous-tag-assisted purification after each reaction cycle allowed the synthesis of short β-mannan oligomers with limited loading of glycosyl donor-as low as 3.0 equivalents for each glycosylation cycle. This study showed the capability of the automated solution-phase synthesis protocol for synthesizing various challenging glycosides, including use of a C-5 ester as a protecting group that could be converted under reductive conditions to a hydroxymethyl group for chain extension.
- Published
- 2016
49. Evaluating Sustainability: Soap versus Biodiesel Production from Plant Oils
- Author
-
Nicola L. B. Pohl, Steve M. Brokman, and Jennifer M. Streff
- Subjects
Green chemistry ,Science instruction ,Rapeseed ,Chemistry ,SOAP ,computer.internet_protocol ,General Chemistry ,Pulp and paper industry ,Education ,Biodiesel production ,Sustainability ,Organic chemistry ,Transesterification reaction ,computer ,Saponification - Abstract
Herein we describe a series of experiments for the undergraduate organic laboratory curriculum in which various plant oils (soybean, rapeseed, and olive) are subjected to saponification and transesterification reactions to create a set of compounds that can function as soaps or as fuels. The experiments introduce students to and asks them to critically evaluate concepts of sustainability and green chemistry, microwave versus conventional heating, and their understanding of chirality–prochirality, and stereochemistry. Unexpectedly, because of the comparison between soap and biodiesel production, the experiment also revealed misunderstandings about catalysis.
- Published
- 2012
50. Mannose-Functionalized 'Pathogen-like' Polyanhydride Nanoparticles Target C-Type Lectin Receptors on Dendritic Cells
- Author
-
Amanda E. Ramer-Tait, Brenda R. Carrillo-Conde, Michael J. Wannemuehler, Nicola L. B. Pohl, Yashdeep Phanse, Ana Vianey Chavez-Santoscoy, Balaji Narasimhan, Eun-Ho Song, and Bryan H. Bellaire
- Subjects
Surface Properties ,media_common.quotation_subject ,T cell ,Pharmaceutical Science ,Mannose ,Bone Marrow Cells ,Receptors, Cell Surface ,chemical and pharmacologic phenomena ,Mice ,chemistry.chemical_compound ,Adjuvants, Immunologic ,C-type lectin ,Polyanhydrides ,Drug Discovery ,medicine ,Animals ,Lectins, C-Type ,CD40 Antigens ,Particle Size ,Receptor ,Internalization ,Cells, Cultured ,media_common ,CD86 ,Histocompatibility Antigens Class II ,Dendritic Cells ,Up-Regulation ,Cell biology ,Mice, Inbred C57BL ,Mannose-Binding Lectins ,medicine.anatomical_structure ,Biochemistry ,chemistry ,Cytokines ,Nanoparticles ,Molecular Medicine ,B7-2 Antigen ,Signal transduction ,Cell Adhesion Molecules ,Mannose Receptor ,Mannose receptor - Abstract
Targeting pathogen recognition receptors on dendritic cells (DCs) offers the advantage of triggering specific signaling pathways to induce a tailored and robust immune response. In this work, we describe a novel approach to targeted antigen delivery by decorating the surface of polyanhydride nanoparticles with specific carbohydrates to provide "pathogen-like" properties that ensure nanoparticles engage C-type lectin receptors on DCs. The surface of polyanhydride nanoparticles was functionalized by covalent linkage of dimannose and lactose residues using an amine-carboxylic acid coupling reaction. Coculture of functionalized nanoparticles with bone marrow-derived DCs significantly increased cell surface expression of MHC II, the T cell costimulatory molecules CD86 and CD40, the C-type lectin receptor CIRE and the mannose receptor CD206 over the nonfunctionalized nanoparticles. Both nonfunctionalized and functionalized nanoparticles were efficiently internalized by DCs, indicating that internalization of functionalized nanoparticles was necessary but not sufficient to activate DCs. Blocking the mannose and CIRE receptors prior to the addition of functionalized nanoparticles to the culture inhibited the increased surface expression of MHC II, CD40 and CD86. Together, these data indicate that engagement of CIRE and the mannose receptor is a key mechanism by which functionalized nanoparticles activate DCs. These studies provide valuable insights into the rational design of targeted nanovaccine platforms to induce robust immune responses and improve vaccine efficacy.
- Published
- 2011
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