Novel, practical chemical reactions for the synthesis of biologically significant molecules as well as development of new tools for studying the structure-function relationship of these carbohydrates are also being investigated in the lab. Carbohydrates of interest include those associated with glycoproteins and those involved in bacterial cell-wall biosynthesis and CD1-mediated T-cell receptor activation. Recent advances in chemical synthesis in our lab include the development of a copper-catalyzed diazotransfer reaction for the conversion of multiple amines to azides; the use of Selectfluor in the synthesis of 2-fluoroglycosides and sugar nucleotides for the mechanistic studies of glycosyltransferases; the discovery of glycosyl phosphites for glycosylation; the large scale synthesis of epothilones; and development of combinatorial chemistry for the synthesis of iminocyclitols and derivatives as glycosyltranfer enzyme inhibitors.
Figure 3A.The proposed mechanism for diazotransfer and its application to the synthesis of perazido neomycin B. The choice of a suitable nitrogen protecting group was critical in the synthesis of aminoglycoside analogs. While alkyl carbamates and trifluoroacetamides have been previously utilized for the protection of primary amines, the presence of multiple Cbz groups makes intrepretation of intermediate NMR spectra difficult. The stability of ethyl and cyclic carbamamtes can also create deprotection and solubility problems during glycosylation. Issues with other amine protection groups included the lability of trifluoroacetamide and Boc groups as well as issues with protection of the pimary acidic NH with acyl-type protecting groups. To solve this crucial issue, we have employed this metal-catalyzed diazotransfer for the facile conversion of amides to azides with streochemical retention. (J. Am. Chem. Soc. (2002) 124, 10773)
Figure 3B. Application of Selectfluor to carbohydrate chemistry. We have found Selectfluor (1-chloromethyl-4-fluoro-1,2-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate), an electrophilic fluorinating agent, to be an extremely selective and potent reagent for the one-pot synthesis of 2-deoxy-2-fluoro glycosides and glycosyl fluorides as well as activating glycosylation reactions. We have also found that the steric constraints of the glycal direct the stereochemistry of fluorine addition. (J. Am. Chem. Soc. (1997) 119, 8146)
We have developed effective chemoenzymatic solid phase synthesis of oligosaccharides, glycopeptides, and their sulfated derivatives and investigated the effect of glycosylation and sulfation on peptide conformation using NMR. We have developed a new enzymatic sulfation method using sulfotransferases coupled with the regeneration of the PAPS cofactor suitable for high throughput assays of sulfotransferases and the large scale enzymatic sulfation reaction. (Angew. Chem. Int. Ed. (2004) 43, 3526)
Figure 3C. Enzymatic sulfation of saccharides with the regeneration of the PAPS cofactor. left: proposed transition state of the reaction. (Angew. Chem. Int. Ed. (1999) 38, 2747)
Figure 3D. Diversity-oriented synthesis of purine derivatives and the nanomolar inhibitor discovered. (J. Am. Chem. Soc. (2002) 124, 14524)
These methods will be used to study the role of sulfate signaling associated with sulfoglycoproteins. We use surface plasmon resonance to determine the binding kinetics and thermodynamics of RNA-small molecule and protein-ligand interactions and use this method to evaluate the activity of our designed inhibitors.
Figure 3E. Development of carbohydrate-based vaccines. We are defining the specificity of CD1-mediated T-cell activation in an effort to develop new glycolipids as vaccines. In addition, hydrophobic glycopeptides are being designed as mucin mimics to stimulate MHC-1 mediated T-cell activation. We are also developing new methods to attach sugar antigens to carrier proteins for development of anti-cancer, anti-HIV and other anti-infective vaccines
We are developing polyvalent glycoconjugates and mimetics to study their interaction with cell-surface receptors or with RNA, and are investigating the effect of these interactions on the expression level of genes and proteins inside the cell using DNA arrays and proteomics, in order to understand how a signal is transduced from the outside to the inside of a cell and what pathway is associated with the signaling. Finally, we are also developing small peptides and nucleic acids enantiomeric to their natural configuration (i.e. D-peptides and L-nucleic acids) as stable and high-affinity receptors of cell-surface carbohydrates using the replicable phage display and nucleic acid selection technologies. The enantiomer of the cell surface carbohydrate, for example, is first synthesized using our chemoenzymatic strategy, and used to screen the a phage or nucleic acid library for binders. After further improvement of the affinity via successive rounds of selection or construction of an oligomer of the binder, the enantiomers which will be chemically synthesized are expected to bind the natural cell-surface carbohydrate target. In addition to development of new drug candidates targeting the cell surface, this approach will provide insights in to the underlying principles of sugar-receptor recognition.
Figure 3F. Development of D-peptides and L-nucleic acid as metabolically stable cell-surface sugar receptors. Our strategy is using our chemo-enzymatic method to prepare the enantiomer of a cell-surface sugar epitope, and use that as an affinity ligand to select small peptides displayed on phage or small DNA or RNA via directed evolution. The enantiomers of these peptides (the D-peptides) or nucleic acids (L-DNA or RNA) should bind the naturally occurring cell-surface sugar. This is also an effective approach to understand the principles of protein-carbohydrate interaction. (ChemBioChem (2001) 2, 741)