Research in our group focuses the development of novel methodologies for the stereoselective synthesis of complex oligosaccharides and the enantioselective construction of nitrogen and fluorine-containing compounds that possess significant bioactivity utilizing transition-metal-catalyzed processes. The bioactive molecules obtained will set the stage for subsequent biological studies as well as the design and development of related structural analogs. Recently, we have developed a rapid and mild conditions for fluorine-18 incorporation into small organic molecules for potential use as PET radiotracers. For each of the above projects, we team up with collaborators at the University of Iowa College of Medicine, the University of Iowa PET Imaging Center, the University of Alabama, Northwestern University, and the University of North Carolina for biological studies.
GLYCOPOLYMER INHIBITION OF HEPARANASE
The carbohydrate-processing enzyme, heparanase, is known to cleave heparan sulfate polysaccharides of the surface of the cell, leading to a cascade of cancer events including tumor growth and metastasis. With the desire to inhibit heparanase, we then utilized our nickel triflate methodology to synthesize the sulfated GlcN-GlcA disaccharide, which sits in the -1/-2 subsite of heparanase. Oligosaccharide studies have presented that heparanase can recognize the disaccharide, however it can not degrade it. We then placed the disaccharide unit onto a multivalent polymer scaffold to enhance the overall protein-saccharide avidity and mimic the natural heparan sulfate structure. The strength of inhibition of heparanase by sulfated 2-aminosugar functionalized polymers were examined in three areas: length of polymer, the display of the saccharides, and the sulfation pattern of the saccharide. From these studies it was determined that the ideal polymer length and saccharide display was a C(6)- and N-sulfated diantennary glycopolymer with a degree of polymerization of 12 repeating units. In a competitive TR-FRET assay, the GlcNS(6S)a(1,4)GlcA glycopolymer had an IC50 = 0.10 ± 0.04 nM towards heparanase. To ensure heparanase specificity, the most potent glycopolymer inhibitor of heparanase was examined with a solution based competitive biolayer interferometry assay for cross-bioactivity to other HS-binding proteins (growth factors, platelet factor 4, P-selectin) which are responsible for mediating angiogenic activity, antibody-induced thrombocytopenia, and tumor cell metastasis. Compared to heparin, our designed synthetic glycopolymer has a much lower affinity for these proteins. Additionally, the synthetic glycopolymer was shown to have antiproliferative properties when analyzed using a HUVEC cell assay and an anti-metastatic effect in a 4T1 mammary carcinoma model.
1) Loka, R. S.;** Sletten, E. T.;** Barash, U.; Vlodavsky, I.; Nguyen, H. M.* “Specific Inhibition of Heparanase by Glycopolymer with Well-Defined Sulfation Pattern Prevents Breast Cancer Metastasis in Mice.” ACS Appl. Mater. Interfaces 2019, 11, 244-254
2) Sletten, E. T.;** Loka, R. S.;** Yu, F.; Nguyen, H. M.* “Glycosidase Inhibitors from Multivalent Presentation of Heparan Sulfate Saccharides on Bottlebrush Polymer.” Biomacromolecules 2017, 18, 3387-3399
3) Loka, R. S.;** Yu, F.;** Sletten, E. T.;** Nguyen, H. M.* “Design, Synthesis, and Evaluation of Heparan Sulfate Mimicking Glycopolymers for Inhibiting Heparanase Activity.” Chem. Commun. 2017, 53, 9163-9166
CATALYTIC STEREOSELECTIVE METHODS FOR 1,2-CIS-2-GLYCOSIDE SYNTHESIS
The stereochemistry of anomeric carbon-oxygen bonds, which is the typical framework of carbohydrate molecules, can have implications for structure and biological function. Consequently, the development of methods that control stereoselective construction of anomeric C(sp3)-O bonds are of paramount importance, but challenging. In general, stereochemical outcomes in anomeric C(sp3)-O bond-forming processes are unpredictable and dependent on the steric and electronic nature of the protecting groups bound on carbohydrate coupling partners. In particular, the stereoselective synthesis of an a-1,2-cis glycoside has proven to be a hurdle when utilizing traditional modes of carbohydrate activation. There have been many strategies developed to address this issue. Unfortunately, current methods often rely on the nature of the protecting groups bound to the electrophile to influence selectivity, thereby making them highly specific for each electrophilic coupling partner. On the other hand, catalytic approaches to alpha-1,2-cis glycosidic bond are relatively limited (7), although they could provide catalyst control without relying on the influence of the protecting group nature of carbohydrate substrates.
A) Phenanthroline-Catalyzed Stereoretentive Glycosylations
In an effort to identify an effective strategy for a stereoselective synthesis of alpha-1,2-cis glycosides that would obviate the necessity for substrate prefunctionalization, we consider whether the anomeric selectivity could be controlled by a simple catalyst. We recently discovered a commercially available phenanthroline to stereoselectively catalyze formation of alpha-1,2-cis glycosides . Phenanthroline is a rigid and planar structure with two fused pyridine rings whose nitrogen atoms are positioned to act cooperatively. We postulated that the first nitrogen atom serves as a catalytic nucleophile to react with an electrophile to generate a covalent beta-glycosyl phenanthrolium ion preferentially as phenanthroline is sterically demanding . The second nitrogen atom could non-covalently interact with carbohydrate moiety or form a hydrogen bond with the alcohol nucleophile to facilitate invertive substitution. These features should effectively promote a double SN2 mechanism. The method proves efficient access to alpha-1,2-cis glycosides and has been performed for the large-scale synthesis of an alpha-1,6-linked octasaccharide adjuvant. Density functional theory calculations, together with kinetic studies, suggest the reaction proceeds via a double SN2 mechanism.
B) Visible-Light Promoted Copper-Catalyzed Stereoselective Glycosylations
Transition-metal catalyzed cross-coupling reactions have become one of the most powerful methods for the construction of organic molecules (9). The highly predictable nature of these cross-coupling reactions had found application in anomeric C(sp3)-C(sp2) and C(sp3)-C(sp3) bond formation to access C-glycosides. However, the implementation of such transformations for stereoselective construction of anomeric C(sp3)-O has been elusive.
Copper has been considered a privileged catalyst because it is an abundant, inexpensive, and relatively non-toxic metal whose high-valent Cu(III) complex has the propensity to undergo facile reductive elimination with a variety of coupling partners (16). Recently, we sought to design a copper-catalyzed cross coupling of an anomeric carbon of alpha-1-bromo glycoside with a hydroxyl of a carbohydrate under excitation by a blue light-emitting diode (LED). We recognized that this light-driven copper catalysis process could result in stereoselective formation of C(sp3)-O glycosidic bond . The successful execution of this concept leads to a general method for stereoselective construction of oligosaccharides bearing a-1,2-cis glycosidic bonds.
NEW METHODS FOR 1,2-CIS-2-AMINOSUGARS AND SYNTHESIS AND BIOLOGICAL STUDIES OF BIOACTIVE OLIGOSACCHARIDES
One facet of our research program involves the development of a series of new methodologies, allowing for the efficient assembly of complex carbohydrate molecules which can be explored as potential therapeutics against cancers, bacterial infections, tuberculosis, and anticoagulation. In particular, we exploit the nature of transition metals as the catalyst to selectively promote the formation of high purity 1,2-cis-2-aminosugars, one of the most important classes of naturally occurring complex carbohydrates and glycoproteins. Progress toward understanding the specific roles that glycoproteins play in biochemical processes is hampered by low isolation yields, inconsistent composition, and the lack of purity of these materials. In many cases, high purity 1,2-cis-2-aminosugars can only be obtained by chemical synthesis. We have developed an innovative method for the synthesis of 1,2-cis-2-aminosugars that utilizes nickel catalysts to direct the coupling. The method relies on the nature of the catalyst rather than protecting groups on the substrate to control the selectivity, is broadly applicable to a wide range of substrates, and provides the coupling products in high yields and with excellent selectivity. The utility of the nickel catalyzed 1,2-cis-2-amino glycosylation method is currently applicable to the synthesis of a number of biologically active complex carbohydrates. Representative molecules include: 1) mycothiol, which is a low molecular weight thiol present in mycobacterium tuberculosis, is a potential target for the development of new treatment against tuberculosis; 2) heparin and heparan sulfate oligomers which prevents blood clotting with minimal side effect and can be used as potent inhibitors of heparanase; 3) heptasaccharide N-linked glycan which is important for the adherence of Campylobacter jejuni to the surface of human host cells and plays a crucial role of the protein glycosylation system in the pathogenesis of C. jejuni; 4) Natural zwitterionic polysaccharide PS A1 has been demonstrated as potential novel vaccine adjuvants. The chemical biology studies of the bioactive oligosaccharides and their corresponding analogues are done in collaboration with Prof. Jon Houtman and Prof. Steve Varga at the University of Iowa College of Medicine and Prof. Jian Liu at UNC-Chapel Hill.
1) McKay, M. J.; Nguyen, H. M. “Recent Advances in Transition Metal-Catalyzed Glycosylation.” ACS Catalysis 2012, 2, 1563-1595.
2) Mensah, E. A.; Yu, F.; Nguyen, H. M. “Nickel-Catalyzed Alpha- Coupling with C(2)-N-Substituted Benzylidene Trichloroacetimidates for the Formation of 1,2-cis-2-Amino Glycosides. Applications to the Synthesis of Heparin Disaccharides, Alpha-GalNAc, and GPI Anchor Pseudodisaccharides.” J. Am. Chem. Soc. 2010, 132, 14288-14302.
3) Mensah, E. A.; Nguyen, H. M. “Nickel-Catalyzed Formation of Alpha-2-Deoxy-2-Amino-Glycosides.” J. Am. Chem. Soc. 2009, 131, 8778-8780.
4) Yu, F.; Nguyen, H. M. “Studies on the Selectivity Between Nickel-Catalyzed 1,2-Cis-2-Amino Glycosylation of Hydroxyl Groups of Thioglycoside Acceptors with C(2)-Substituted Benzylidene N-Phenyl Trifluoroacetimidates and Intermolecular Aglycon Transfer of the Sulfide Group.” J. Org. Chem. 2012, 77, 7330-7343.
5) McConnell, M. S.; Yu, F.; Nguyen, H. M. “Nickel-Catalyzed alpha-Glycosylation of C(1)-Hydroxyl Group of Inositol Acceptors: A Formal Synthesis of Mycothiol.” Chem. Commun. 2013, 49, 4313-4315.
6) Yu, F.; McConnell, M. S.; Nguyen, H. M. “Scalable Synthesis of Fmoc-Protected GalNAc-Threonine Amino Acid and TN Antigen via Nickel Catalysis.” Org. Lett. 2015, 17, 2018-2021.
7) Sletten, E. T.; Ramadugu, S. K.; Nguyen, H. M. “Utilization of Bench-Stable and Readily Available Nickel (II) Triflate for Access to 1,2-Cis-2-Aminoglycosides.“ Carbohydrate Research 2016, 435, 195-207.
8) Sletten, E. T.; Tu, Y.-T.; Schlegel, H. B.;* Nguyen, H. M.* “Are Brønsted Acids the True Promoter of Metal Triflate Catalyzed Glycosylations? A Mechanistic Probe into 1,2-cis-Aminoglycoside Formation by Nickel Triflate.” ACS. Catalysis 2019, 9, 2110-2123.
NITROGEN-SUBSTITUTED QUARTERNARY CENTERS
Many practical and elegant routes to the synthesis of amine-containing compounds have been developed. There are, however, limited approaches available for the asymmetric synthesis of alpha, alpha-disubstituted amines, the class of nitrogen-substituted quaternary centers. Transition metal-catalyzed substitution of primary and secondary allylic carbonates or acetates has been utilized for the preparation of chiral alpha-substituted N-arylamines. We have developed the dynamic kinetic asymmetric transformations (DYKAT) of racemic tertiary allylic imidates with anilines utilizing Hayashi’s bicyclo[2.2.2]-octadiene ligand-ligated rhodium catalyst. The method allows for the preparation of alpha, alpha-disubstituted allylic N-arylamines in good yields and with excellent regio- and enantioselectivity. The ability of the rhodium-catalyzed DYKAT of racemic tertiary allylic imidates is currently applying to the synthesis of a number of bioactive natural products. Representative targets includes: 1) myriocin, which is one of the most potent immunosuppressant natural products, inhibits serine palmitoyltransferase, an enzyme in the biosynthesis of sphingolipid; 2) manzacidins exhibit useful activities as alpha-adrenoreceptor blockers, actomyosin ATPase activators, and serotonin antagonists. However, these compounds have only been preliminarily tested due to their limited quantities from natural sources; 3) BIRT-377, a potent inhibitor of cell adhesion molecule-1(ICAM-1) and lymphocyte function-associated antigen. Thus, BIRT-377 could play a vital function in the treatment of various inflammatory and immune disorders, and 4) pactamycin, a highly potent antitumor and antibacterial antibiotic isolated from Streptomyces pactum var. pactum. Jogyamycin and TM-026 were isolated via biosynthesis and genetic engineering studies. While TM-026 exhibited potent antimalarial activity, jogyamycin displayed antibacterial activity.
1) Arnold, J. A.; Stone, R. F.; Nguyen, H. M. “Rhodium-Catalyzed Regioselective Amination of Secondary Allylic Trichloroacetimidates with Aromatic Amines.” Org. Lett. 2010, 12, 45804583.
2) Arnold, J. S.; Cizio, G. T.; Nguyen, H. M. “Synthesis of a,a-Disubstituted Allylic Aryl Amines by Rhodium-Catalyzed Amination of Tertiary Allyic Imidates.” Org. Lett. 2011, 13, 5576-5578.
3) Arnold, J. S.; Nguyen, H. M. “Rh-Catalyzed Dynamic Kinetic Asymmetric Transformations of Racemic Tertiary Allylic Imidates with Anilines.” J. Am. Chem. Soc. 2012, 134, 8380-8383.
4) Arnold, J. S.; Cizio, G. T.; Heitz, D. R.; Nguyen, H. M. “Rhodium-Catalyzed Regio- and Enantioselective Amination of Racemic Secondary Allylic Imidates with N-Methyl Anilines.” Chem. Commun. 2012, 48, 11531-11533.
5) Arnold, J. S.; Nguyen, H. M. “Rhodium-Catalyzed Enantioselective Amination of Allylic Trichloroacetimidates.” Synthesis 2013, 45, 2101-2108.
6) Arnold, J. S.; Mwenda, E. E. T.; Nguyen, H. M. “Sequential Amination and Hydroacylation Reactions for the Enantioselective Synthesis of Seven-Membered Heterocycles.” Angew. Chem. Int. Ed. 2014, 53, 3688-3692.
7.) Mwenda, E. T.; Nguyen, H. M. “Enantioselective Synthesis of 1,2-Diamines Containing Tertiary and Quaternary Centers through Rodium-Catalyzed DYKAT of Racemic Allylic Trichloroacetimidates” Org. Lett. 2017, 19, 4814-4817.
FLUORINE-CONTAINING COMPOUNDS FOR PET IMAGING
Fluorine-containing compounds play a prominent role in positron emission tomography (PET) imaging, one of the most rapidly growing areas of non-invasive medical imaging. Currently, PET has been widely used in diagnosis of cancers, cardiovascular diseases, early stage neurological disorders and in clinical trials. Measuring physiochemical processes with PET requires imaging agents labeled with positron-emitting radioisotopes. Fluorine-18 (18F) appears to be the most ideal isotope for PET imaging due to its low energy positron emission, ease of preparation, and ideal half life (110 min). The incorporation of fluorine into organic molecules by direct carbon-fluorine bond formation has proven to be quite challenging. We utilize trichloroacetimidates in conjunction with an iridium catalyst and triethylamine-hydrofluoride (Et3N.3HF) reagent to provide allylic fluorides in good yields and with excellent selectivity. The reaction is conducted at room temperature under ambient air and shows considerable functional group tolerance. Use of potassium fluoride-kryptofix complex, the most commonly used source of 18F-fluoride ion in PET imaging, allows 18F-incorporation into allylic electrophiles in 10 minutes. We continue our efforts by developing new strategies which allow rapid and efficient formation of benzylic and alpha-branching allylic C-F bonds. These proposed efforts will deliver suitable synthetic methods for the enantioselective synthesis of alpha-branching heterocyclic and beta-/gama-amino substituted allylic fluorides and the rapid access to [18F]fluorine-containing molecules. The methodologies have potential application in the enantioselective synthesis of potent and selective fluorinated neuronal nitric oxide synthase (nNOs) inhibitors for use in treatment of neurodegenerative diseases and in the preparation of [18F]-containing nNOS inhibitors for utilization as PET radiotracers to monitor Alzheimer’s, Parkinson’s, Huntington’s disease. This project is done in collaboration with Prof. David Dick at the University of Iowa PET Imaging Center and Prof. Silverman at Northwestern University.
1) Topczewski, J. J.; Tewson, T. J.; Nguyen, H. M. “Iridium-Catalyzed Allylic Fluorination of Trichloroacetimidates.” J. Am. Chem. Soc. 2011, 133, 19318-19321.
2) Zhang, Q.; Nguyen, H. M. “Rhodium-Catalyzed Regioselective Ring Opening of Vinyl Epoxides with Et3N.3HF: Formation of Allylic Fluorohydrins.” Chem. Sci. 2014, 5, 291-296.
3) Zhang, Q; Mixdorf, J. C.; Reynders, G. J.; Nguyen, H. M. “Rhodium-Catalyzed Benzylic Fluorination of Trichloroacetimidates with Et3N.3HF.” Tetrahedron. 2015, 71, 5932-5938.
4) Zhang, Q; Stockdale, D. P.; Mixdorf, J. C.; Topczewski, J. J.; Nguyen, H. M. “Iridium-Catalyzed Enantioselective Fluorination of Racemic, Secondary Trichloroacetimidates.” J. Am. Chem. Soc. 2015, 137, 11912-11915.
5) Mixdorf, J. M.; Sorlin, A. M.; Nguyen, H. M. “Asymmetric Synthesis of Allylic Fluorides via Fluorination of Racemic Allylic Trichloroacetimidates Catalyzed by a Chiral Diene-Iridium Complex.” ACS Catal., 2018, 8, 790-801.
6) Mixdorf, J. C;** Sorlin, A. M.;** Dick, D.;* Nguyen, H. M.* “Iridium-Catalyzed Radiosynthesis of Branched Allylic [18F]Fluorides.” Org. Lett. 2019, 21, 60-64.