Project 1: Exploring the Nature of Ligand Binding in the Active Site of Galectin-1 Using Natural and Unnatural Carbohydrates
Human galectin-1 (Figure 4) is expressed on cell surfaces and in extra cellular matrices, and is involved in a number of critical processes including inflammation, development, mRNA splicing, differentiation, and cell adhesion. [1]-[2] In normal cells the expression of galectin-1 is regulated. Diseased or stressed cells have been shown to over express galectin-1. For example, galectin-1 has been found in unusually high concentrations in and around tumor cells and has been implicated in several aspects of cancer biology including tumor transformation,[3] apoptosis,[4] cell growth regulation,[5] and metastasis.[6] Research has also shown that galectin-1 may play an important role in protecting tumor cells from immune attack,[7] and studies have also suggested that galectin-1 may play an important role in the promotion of HIV infectivity.[8]
Figure 4: Structure of the galectin-1 bound to lactose (Taken from Camby et. al.).
Notes: Amino acids highlighted in green illustrate highly conserved residues. Amino acid residues highlighted in pink are known to interact with bound carbohydrates via hydrogen bonding interactions. Amino acids highlighted in orange are known to interact with bound carbohydrates via vDW forces.
Because of the biological role galectin-1 plays in a number of diseases, considerable research has been devoted to the design and synthesis of specific galectin-1 inhibitors. Despite recent advances in the preparation and evaluation ligands that target and bind galectin-1, little is known about the nature of the galectin-1 ligand binding interaction. Students in my laboratory will prepare natural and unnatural carbohydrate triazoles that mimic poly-N-acetyllactosamine, the natural ligand for galectin-1 (Figure 5). The proposed derivatives will be used to further an understanding of galectin-1-ligand binding, and the knowledge gained will serve as a foundation for the design and preparation of agents that selectively target galectin-1. The derivatives incorporating unnatural carbohydrates are especially interesting as studies suggest that the increased flexibility should allow the mimetic to bind preferentially over the natural ligand thus inhibiting galectin-1.
Figure 5: Potential galectin-1 inhibitors.
Project 2: Synthesis of a Vancomycin Derivative Incorporating an Unnatural Carbohydrate at the Vancosamine Position
Vancomycin (Figure 6A) is a broad spectrum glycopeptide antibiotic that is generally used as a “last resort” for the treatment of gram positive bacterial infections, such as those caused by staphylococcus (“staph” infections). Vancomycin is composed of two bioactive components, a cyclic peptide component called an aglycon, and a functionalized peripheral carbohydrate component called a glycan. These two units work together to blocking the approach of several key enzymes involved in bacteria cell wall biosynthesis.[9]
The role of the aglycon in the inhibition of peptidoglycan biosynthesis is well understood. The aglycon, forms a binding pocket in which five key amino acid residues in the pocket hydrogen bond to peptidoglycan precursors terminating in the amino acid sequence D-alanyl-D-alanine (D-Ala-D-Ala).[10] Once bound, the aglycon creates an obstruction that impedes the processing of immature precursors into mature petidoglycan. The role of the glycan, however, is less understood. Experimental evidence has shown that the glycan is involved in several key recognition events that serve to define vancomycin’s overall structural integrity and biological activity. The glycan is believed to play an important role in the conformational maintenance of the aglycon and assists the aglycon in dimerization and membrane anchoring events. [11] These events are believed to increase the binding avidity of the antibiotic. In addition, recent research has suggested that the glycan may be involved in the direct inhibition of the transglycosylases involved in transglycosylation, regardless of a petidoglycan binding event.[12]
Over the past twenty years, several vancomycin-resistant strains of bacteria have been detected.[13] This has led researchers to search for new and more potent derivatives of vancomycin. Students in my research group will work together to prepare a new derivative of vancomycin (Figure 6B) that incorporates an unnatural (septanose) residue at the vancosamine position of the glycan. Previous studies involving septanose carbohydrates suggest that they may require less energy than their natural (pyranose) counterparts to bind to specific enzymes involved in bacterial cell wall biosynthesis. In addition, the unnatural nature of the septanose carbohydrate makes it more difficult for the bacteria to develop a resistance to the analog.
Figure 6: Vancomycin (A) and a vancomycin derivative incorporating a septanose residue (B).
The derivative prepared in this study will be assessed for activity against a panel of vancomycin-resistant bacteria, including Enterococci (E. faecium and E. faecalis) and Staphylococci (methicillin resistant strains--MRSA). The results from this study will be used to further an understanding of the role of the carbohydrate component of vancomycin in fighting gram positive bacteria, and the knowledge gained here will serve as a foundation for the design and preparation of other glycopeptide antibiotic for which resistant strains of bacteria have developed.
Project 3: Synthesis and Evaluation of Carbophyrins as Catalysts in Asymmetric Transfer Reactions.
Chiral cyclopropane rings are found in a number of biologically relevant natural products. For example, the antifungal nucleoside 7-A,[14] antitumor curacin A 7-B,[15] and antifungal ambruticin 7-C[16] all contain chiral cyclopropane units that are critical to the biological functions of these compounds. Because of the importance of chrial cyclopropane rings, a number of reactions have been developed for their synthesis.[17] However, the development of catalysts that can catalyze asymmetric cyclopropanation with a variety of substrates in high yield and with excellent diastereo- and enantioselectivity is still a major area of research in the field.
Figure 7: Examples of biologically relevant natural products containing cyclopropane rings.
The recent development of palladium-catalyzed cross-coupling reactions between mono-, di-, and tetrasubstituted bromo-porphyrins and amides, amines, alcohols, and thiols has allowed for the preparation of a number of chiral amino-, amido-, oxo- and mercaptoporphyrins.[18] The cobalt derivatives of these chiral porpyrins have, in turn, been used to catalyze a number of key functional group transformations including the asymmetric cyclopropanation of aromatic and electron-deficient olefins using diazo reagents with good diastereo- and enantioselectivity.[19] Despite the progress that has been made in the development of these catalysts, two key problems still exist. First, the hydrophobic nature of the porphyrin ring makes these systems insoluble in most polar solvents. This limits the utility of these catalysts under polar reaction conditions, further limiting the types of substrates that can be cyclopropanated. In addition, the aromatic nature of the porphyrin ring system facilitates pi stacking with some existing porphyrin catalysts. This results in catalyst aggregation and decreased turnover.
In an effort to address these problems, my undergraduate students and I have started to synthesize novel porphyrins bearing carbohydrate residues in collaboration with Dr. Peter Zhang’s group at the University of South Florida. Carbohydrate-porphyrin conjugates or “CarboPhyrins,” are chiral molecules that have the potential to serve as asymmetric catalysts. In addition to conferring chirality, we believe the carbohydrate ligands will help improve the overall solubility of the porphyrin catalysts in polar solvents, including water. The conformational nature of the carbohydrate ring should also help decrease the pi stacking effects observed in some systems, without sacrificing stereoselectivity and yield.
The carbohydrate-porphyrin conjugates we have synthesized thus far have been prepared by cross coupling bromo-porphyrin synthons with selectively functionalized carbohydrates using tris-(dibenzylideneacetone)-dipalladium(0) (Pd2(dba)3) as a source of palladium and bis(2-dipheneylphosphinophenyl) ether (DPEphos) as a ligand source in the presence of cesium carbonate (Cs2CO3). Through these preliminary studies, we were able to assess the feasibility of performing palladium catalyzed cross coupling reactions with glucose via different carbohydrate linkages. The results obtained from these experiments have provided us with a foundation for the preparation of additional carbohydrate-porphyrin conjugates containing multiple carbohydrate ligands that can serve as effective and efficient catalyst for the asymmetric synthesis of cyclopropane rings. Examples of these carbohydrate porphyrin conjugates can be seen in Figure 8 below.
Figure 8: Sample carbohydrate-porphyrin conjugates.
The complexes prepared in this study will be evaluated by Hamilton College undergraduates for their ability to serve as catalysts in asymmetric cyclopropanation reactions. Future studies will involve the preparation and evaluation of additional carbohydrate-porphyrin conjugates based on the results obtained from this study.
References
[1] Leffler, H.; Carlsson, S.; Hedlund, M.; Qian, Y.; Poirier, F. “Introduction to Galectins.” Glycoconj. J. 2004, 19, 433.
[2] (a) For recent reviews see Rabinovich, G. A. “Galectin-1 As a Potential Cancer Target.” Br. J. Cancer. 2005, 92, 1188-1192. (b) Camby, I.; Mercier, M. L.; Lefranc, F.; Kiss, R. “Galectin-1: A Small Protein with Major Functions.” Glycobiology 2006, 16, 137-157..
[3] Paz, A.; Haklai, R.; Elad-Sfadia, G.; Ballan, E.; Kloog, Y. „Galectin-1 Binds Oncogenic H-Ras to Mediate Ras Membrane Anchorage and Cell Transformation.” Oncogene 2001, 20, 7486-7493.
[4] (a) Perillo, N. L.; Pace, K. E.; Seilhamer, J. J.; Baum, L. G. “Apoptosis of T-cells Mediated by Galectin-1.” Nature 1995, 378, 736-739. (b) Rabinovich, G. A.; Ramhorst, R. E.’ Rubinstein, N.; Corigliano, A.; Daroqui, M. C.; Kier-Joffe, E. B.; Fainboim, L. “Induction of Allogenic T-cell Hyporesponsiveness to Galectin-1 Mediated Apoptotic and Non-Apoptotic Mechanisms.” Cell Death Diff. 2002, 9, 661-670.
[5] (a) Wells, V.; Davies, D.; Mallucci, L. “Cell Cycle Arrest and Induction of Apoptosis by β-Galactoside-Binding Protein (b-GBP) in Human Mammary Cancer Cells.” Eur. J. Cancer 1999, 35, 978-983. (b) Yamaoka, K.; Mishima, K.; Nagashima, Y.; Asai, A.; Sanai, Y.; Kirino, T. J. “Expression of Galcetin-1 mRNA Correlates with the Malignant Potential of Human Gliomas and Expression of Antisense Galectin-1 Inhibits The Growth of 9 Glioma Cells.” Neurosci. Res. 2000, 59, 722-730. (c) Kopitz, J.; von Reizenstein, C.; Andre, S.; Kaltner, J.; Uhl, J.; Ehemann, V.; Cantz, M.; Gabius, H.-J. “Negative Regulation of Neuroblastoma Cell Growth by Carbohydrate Dependaant Surface Binding of Galectin-1 and Functional Divergence from Galectin-3.” J. Biol. Chem. 2001, 276, 35917-35923.
[6] (a) Ellerhorst, J.; Nguyen, T.; Cooper, D. N.; Lotan, D.; Lotan, R. “Differential Expression of Endogenous Galectin-1 and Galectin-3 in Human Prostate Cancer Cell Lines and Effects of Overexpressing Galectin -1- on Cell Phenotype.” Int. J. Oncol. 1999, 14, 217-224. (b) van den Brule, F.; Califice, S.; Castronovo, V. “Expressions of Galectins in Cancer: A Critical Review.” Glycoconj. J. 2004, 19, 537-542. (c) Tinari, N.; Kuwabara, I.; Hufflejt, M. E.; Shen, P. F.; Iacobelli, S.; Liu, F.-T. “Glycoprotein 90K/MAC-2BP Interacts with Galcetin-1 and Mediates Galcetin-1-Induced Cell Aggregation.” Int. J. Cancer 2001, 91, 167-172. (d) Hittelet, A.; Legender. H.; Nagy, N.; Bronckart, Y.; Pector, J. C.; Salmon. I.; Yeaton, P.; Gabius, H. J.; Kiss, R.; Camby, I. “Upregulation of Galectins-1 and -3 in Human Colon Cancer and Their Role in Regulation Cell Migration.” Int. J. Cancer 2003, 103, 370-379. (d) Clausse, N.; van den Brule, F.; Waltregny, D.; Garnier, F.; Castronovo, V. “Galectin-1 Expression in Prostate Tumor Associated Capillary Endothelial Cells is Increased by Prostate Carcinoma Cells and Modulates Heterotypic Cell-Cell Adhesion.” Angiogenesis 1999, 3, 317-325. (e) Rabinovich, G. A.; Rubinstein, N.; Matar, P.; Rozados, V.; Gervasoni, S.; Scharovsky, G. O. “The Antimetastatic Effect of a Single Low Dose of Cylophosphamide Involves Modulation of Galcetin-1 and Bcl-2 Expression.” Cancer Immunol. Immunother. 2002, 19, 597-603.
[7] He, J.; Baum, L.G. “Presentation of Galectin-1 by Extracellular Matrix Triggers T Cell Death.” J. Biol. Chem. 2004, 279, 4705-4712.
[8] Ouellet, M.; Mercier, S.; Pelletier, I.; Bounou, S.; Roy, J.; Hirabayashi, J.; Sato, S.; Tremblay, M. J. “Galectin-1 Acts as a Soluble Host Factor That Promotes HIV-1 Infectivity through Stabilization of Virus Attachment to Host Cells.” J. Immunol. 2005, 174, 4120-4126.
[9] Gale, E. F.; Cundliffe, E.; Reynolds, P.; Richmond, M. H.; Warning, M. J. “The Molecular Basis of Antibiotic Action.” Wiley-Interscience, New York, 1981.
[10] Perkins, H. R. “Specificity of Combination Between Mucopeptide Precursors and Vancomycin or Ristocetin.” Biochem J. 1969, 111, 195.
[11] Sharman, G. J.; Try, A. C.; Dancer, R. J.; Cho, Y. R.; Staroske, T.; Bardsley, B.; Maguire, A. J.; Cooper, M. A.; O'Brien, D. P.; Williams, D. H. “The Roles of Dimerization and Membrane Anchoring in Activity of Glycopeptide Antibiotics Against Vancomycin-Resistant Bacteria ”JACS, 1997, 119, 12041.
[12] Ge, M.; Chen, Z.; Onishi, R.; Kohler, J.; Silver, L.; Kerns, R.; Fukuzawa, S.; Thompson, C.; Kahne, D. “Vancomycin Derivatives That Inhibit Peptiodglycan Biosynthesis Without Biding D-Ala-D-Ala.” Science 1999, 284 (16) 507.
[13] (a) Bugg, T. D. H.; Dutka,-Malen, S.; Arthur, P.; Courvalin, P.; Walsh, C. T. “Identification of Vancomycin Resistance Protein VanA as a D-alanine:D-alanine Ligase of Altered Substrate Specificity” Biochemistry 1991, 30, 2017. (b) Arthur, M.; Courvalin, P. “Genetics and Mechanisms of Glycopeptide Resistance in Enterococci.” Antimicrob Agents Chemother. 1993, 37, 1563. (c) Walsh, C. T. “"Vancomycin Resistance: Decoding the Molecular Logic." Science 1993, 261, 308.
[14] (a) Barrett, A. G. M.; Kasdorf, K. “Total Synthesis of the Pentacyclopropane Antifungal Agent FR-900848.” J. Am. Chem. Soc. 1996, 118, 11030-7. (b) Barrett, A. G. M.; Doubleday, W. W.; Kasdorf, K.; Tustin, G. J. “Stereochemical Elucidation of the Pentacyclopropane Antifungal Agent FR-900848.” J. Org. Chem. 1996, 61, 3280-8. (c) Yoshida, M.; Ezaki, M.; Hashimoto, M.; Yamashita, M.; Shigematsu, N.; Okuhara, M.; Kohsaka, M.; Horikoshi, K. “A novel antifungal antibiotic, FR-900848. I. Production, isolation, physico-chemical and biological properties.” J. Antibiot. 1990, 43, 748-54.
[15] (a) Barrett, A. G. M.; Kasdorf, K. “Total Synthesis of the Pentacyclopropane Antifungal Agent FR-900848.” J. Am. Chem. Soc. 1996, 118, 11030-7. (b) Barrett, A. G. M.; Doubleday, W. W.; Kasdorf, K.; Tustin, G. J. “Stereochemical Elucidation of the Pentacyclopropane Antifungal Agent FR-900848.” J. Org. Chem. 1996, 61, 3280-8. (c) Yoshida, M.; Ezaki, M.; Hashimoto, M.; Yamashita, M.; Shigematsu, N.; Okuhara, M.; Kohsaka, M.; Horikoshi, K. “A novel antifungal antibiotic, FR-900848. I. Production, isolation, physico-chemical and biological properties.” J. Antibiot. 1990, 43, 748-54.
[16] (a) Kende, A. S.; Mendoza, J. S.; Fujii, Y. “Total synthesis of natural (+)-ambruticin.” Tetrahedron 1993, 49, 8015-38. (b) Kende, A. S.; Fujii, Y.; Mendoza, J. S. “Total synthesis of natural ambruticin.” J. Am. Chem. Soc. 1990, 112, 9645-6. (c) Ringel S. M.; Greenough R. C.; Roemer S.; Connor D.; Gutt A. L.; Blair B.; Kanter G.; von Strandtmann M. “Ambruticin (W7783), a new antifungal antibiotic.” J. Antibiot. 1977, 30, 371-5. (d) Connor, D. T.; Greenough, R. C.; von Strandtmann, M. “W-7783, a unique antifungal antibiotic.” J. Org. Chem. 1977, 42, 3664-9.
[17] For recent reviews see: (a) Nicolas, I.; Le Maux, P.; Simonneaux, G. “Asymmetric catalytic cyclopropanation reactions in water.” Coord. Chem. Rev. 2008, 252, 727-35. (b) Pellissier, H. “Recent developments in asymmetric cyclopropanation.” Tetrahedron 2008, 64, 7041-95. (c) Donaldson, W. A. “Synthesis of cyclopropane containing natural products.” Tetrahedron 2001, 57, 8589-8627.
[18] For select examples see: (a) Gao G.-Y.; Ruppel J. V.; Fields K. B.; Xu X.; Chen Y.; Zhang X P. “Synthesis of diporphyrins via palladium-catalyzed C-O bond formation: effective access to chiral diporphyrins.” J. Org. Chem. 2008, 73, 4855-8. (b) Gao, G.-Y.; Ruppel, J. V.; Allen, D. B.; Chen, Y.; Zhang, X. P. “Synthesis of β-functionalized porphyrins via palladium-catalyzed carbon-heteroatom bond formations: expedient entry into β -chiral porphyrins. J. Org. Chem. 2007, 72, 9060-66. (c) Chen, Y.; Gao, G.-Y.; Zhang, X. P. “Palladium-mediated synthesis of novel meso-chrial porphyrins for cobalt catalyzed cyclopropanation.” Synthesis 2006, 10, 1697-1700. (d) Chen, Y.; Fields, K. B.; Zhang, X. P. “Bromoporphyrins as versatile synthons for modular construction of chiral porphyrins: cobalt-catalyzed highly enantioselective and diastereoselective cyclopropanation.” J. Am. Chem. Soc. 2004, 126, 14718. (e) Gao, G.-Y.; Chen, Y.; Zhang, X. P. “General synthesis of meso-amidoporphyrins via palladium-catalyzed amidation. Org. Lett. 2004, 6, 1837-40. (e) Gao, G.-Y.; Colvin, A. J.; Chen, Y.; Zhang, X. P. “Synthesis of meso-arylsulfanyl- and alkylsulfanyl-substituted porphyrins via palladium-mediated C-S bond formation.” J. Org. Chem. 2004, 69, 8886-92. (f) Chen, Ying; Zhang, X. P. “Facile and efficient synthesis of meso-arylamino- and alkylamino-substituted porphyrins via palladium-catalyzed amination.” J. Org. Chem. 2003, 68, 4432-38. (g) Gao, G.- Y.; C., Yi.; Zhang, X. P. “General and efficient synthesis of arylamino- and alkylamino-substituted diphenylporphyrins and tetraphenylporphyrins via palladium-catalyzed multiple amination reactions.” J. Org. Chem. 2003, 68, 6215-21. (h) Gao, G.-Y.; Colvin, A. J.; Chen, Y.; Zhang, X. P. “Versatile Synthesis of meso-aryloxy- and alkoxy-substituted porphyrins via palladium-catalyzed C-O cross-coupling reactions. Org. Lett. 2003, 5, 3261-64.
[19] For examples see: 5c and (a) Zhu, S.; Perman, J. A.; Zhang, X. P. “Acceptor/acceptor-substituted diazo reagents for carbene transfers: cobalt-catalyzed asymmetric Z-cyclopropanation of alkenes with α -nitrodiazoacetates.” Angew. Chem., Intl. Ed. 2008, 47, 8460-63. (b) Zhu, S.; Ruppel, J. V.; Lu, H.; Wojtas, L.; Zhang, X. P.. “Cobalt-catalyzed asymmetric cyclopropanation with diazosulfones: rigidification and polarization of ligand chiral environment via hydrogen bonding and cyclization.” J. Am. Chem. Soc. 2008, 130, 5042-43. (c) Chen, Y.; Ruppel, J. V.; Zhang, X. P. “Cobalt-catalyzed asymmetric cyclopropanation of electron-deficient olefins.” J. Am. Chem. Soc. 2007, 129, 12074-75. (d) Chen, Y.; Zhang, X. P. “Asymmetric cyclopropanation of styrenes catalyzed by metal complexes of D2-symmetrical chiral porphyrin: Superiority of cobalt over iron.” J. Org. Chem. 2007, 72, 5931-34. (e) Huang, L.; Chen, Y.; Gao, G.-Y.; Zhang, X. P. “Diastereoselective and enantioselective cyclopropanation of alkenes catalyzed by cobalt porphyrins.” J. Org. Chem. 2003, 68, 8179-84.
Note: The left image in the mast head on this page was adapted from Camby, I.; Mercier, M. L.; Lefranc, F.; Kiss, R. “Galectin-1: A Small Protein with Major Functions.” Glycobiology 2006, 16, 137-157.. The right image in the mast head on this page was generated using the glycosyltransferase-inhibitor overlay by published on the web by Strynadka et. al. in “Bacterial Walls Come Tumbling Down.” HHMI Press Release PR-HHMI-07-3, published on the web March 09, 2007.).
Publications:
Fields, K. B.; Ruppel, J. V.; Snyder, N. L.; Zhang, X. P. "Porphyrin Functionalizaton via Palladium-Catalyzed Carbon–Heteroatom Cross-Coupling Reactions." In The Porphyrin Science Handbook; Kadish, K.; Smith, K.; Guillard, R. Eds. (Invited Chapter—Submitted)
Ruppel, J. V.; Fields, K. B.; Snyder, N. L.; Zhang, X. P. "Metalloporphyrin-Catalyzed Asymmetric Atom/Group Transfer Reactions." In The Porphyrin Science Handbook; Kadish, K.; Smith, K. M.; Guillard, R. Eds. (Invited Chapter—Submitted)
Ruppel, J. V.; Gauthier, T. J.; Perman, J.A.; Snyder, N.L.; Zhang, X.P. "Asymmetric Cobalt-Catalyzed Cyclopropanation with Succinimidyl Diazoacetate: General Synthesis of Optically Active Cyclopropyl Carboxamides." Organic Letters 2009, 11, 2273-2276.
Markad, S.D; Xia, S.; Snyder, N.L.; Hadad, C. M.; Peczuh, M. W. “Stereoselectivity in the Epoxidation of Carbohydrate Based Oxepines.” Journal of Organic Chemistry 2008, 73(16), 6341-6354.
Castro, S.; Cherney, E. C.; Snyder, N. L.; Peczuh, M. W. “Synthesis of Substituted Septanosyl-1,2,3-triazoles.” Carbohydr. Res. 2007, 342, 1366-1372.
Snyder, N.L.; Peczuh, M.W. Haines, H.M. “Recent Developments in the Synthesis of Oxepines.” Tetrahedron 2006, 62, 9301-9320
Castro, S.; Duff, M.; Snyder, N.L.; Morton, M.; Kumar, C.V.; Peczuh, M.W. “Recognition of Ring Expanded Carbohydrates by Concanavalin A: The Effect of Anomeric Configuration on Binding.” Org. Biomol. Chem. 2005, 3, 3869-3872.
DeMatteo, M. P.; Snyder N. L; Morton, M.; Baldisseri, D.; Haddad, C.M.; Peczuh, M.W. "Septanose Carbohydrates: Synthesis and Conformational Studies of Methyl a-D-Glycero-D-idoseptanoside and Methyl-b-D-glycero-D-guloseptanosdie." J. Org. Chem. 2005, 70, 24-38.
Peczuh, M.W.; Snyder, N.L.; Fyvie, W.S. “Synthesis, Crystal Structure, and Reactivity of a D-xylose Based Oxepine.” Carbohydr. Res. 2004, 339(6), 1163-1171
Peczuh, M.W.; Snyder, N.L. “Septanals: Ring Expanded Glycals for the Synthesis of Septanose Carbohydrates.” Tetrahedron Lett. 2003, 44, 4057-4061.
