The Skaggs Institute
for Chemical Biology

Scientific Report 2006

Viruses and Click Chemistry for Construction of Functional Nanoparticles

M.G. Finn, E. Kaltgrad, D. Prasuhn, V. Rodionov, E. Strable

Support from the Skaggs Institute has been crucial in the further development of virus particles as platforms for the display of biologically active structures. Three projects deserve special mention.

Mechanism of the Azide-Alkyne Cycloaddition Reaction

We have continued to develop the copper-catalyzed azide-alkyne cycloaddition reaction for bioconjugation; this process is now used by many laboratories around the world. However, further improvements are necessary to tackle the toughest problems of biomolecular construction. To achieve those improvements, we must understand in greater detail the mechanism of the reaction. In the first phase of these studies, we explored new sets of ligands that accelerate the reaction and determined the properties of the reaction involving those ligands. Improved conditions of buffer, catalyst concentration, and reducing agent and the outline of the reaction mechanism have been identified.

Virus-Carbohydrate Conjugates as Potential Vaccines

Carbohydrates are normally invisible to the immune system because of their natural presence in high concentration throughout the tissues. We previously showed that carbohydrates can be made antigenic when arrayed on viral capsids as carrier proteins, consistent with the hypothesis that attachment to the regular polyvalent surfaces of viruses allows the immune system to recognize the displayed structures as foreign. We have focused on the production of IgY antibodies in chickens, because these antibodies can be isolated in large quantities and are highly stable and bind tightly. In the past year, we completed a preliminary study of this process and found that highly selective antibody responses can be generated against several carbohydrates of biological importance. Figure 1 shows the structures involved, which either bind to receptors that are strongly upregulated in cancer cells, are produced in quantity exclusively by cancer cells, or are important in mediating adhesion of pathogens to target tissues. In each instance, the development of antibodies to the carbohydrate structures would be of great importance for diagnostic or therapeutic purposes. An example of the specificity of carbohydrate binding by the resulting antibodies is shown at the bottom of the Figure. These developments have been used by the Consortium for Functional Glycomics, which will disseminate the antibodies we make to interested laboratories within and external to Scripps Research.

Fig. 1. Top, Structures of 4 carbohydrate-azides attached to cowpea mosaic virus and bacteriophage Qβ for immunogenicity studies in chickens. Bottom, Determination of the binding specificity of polyclonal IgY antibodies against the blood group A antigen attached to cowpea mosaic virus. Each vertical line represents the relative intensity of the binding of the antibodies to an individual carbohydrate arrayed on a glass slide. The results show a strong and specific response to blood group A and B antigens that is comparable to the performance that would be exhibited by a monoclonal antibody that is much more expensive and time-consuming to prepare.

We also have had exciting preliminary results with virus particles covered with the carbohydrates rich in mannose that make up the defensive coating of the virus that causes AIDS. When we array these carbohydrates on our innocuous virus carriers, the resulting particles can interfere with the binding of HIV to an antibody currently known to be protective against viral infection when present in high concentrations in the body. Our virus-carbohydrate particles are therefore of interest as potential vaccines, and preliminary experiments in rabbits are under way in collaboration with D.R. Burton at Scripps Research.

New Chemically Modified Viruses

We have found bacteriophage Qβ is a superior platform for the incorporation of unnatural amino acids and their subsequent derivatization by the azide-alkyne cycloaddition reaction. This particle now forms the basis of the immunization studies previously described, as well as other efforts in the areas of cancer targeting and catalysis.

Publications

Dìaz, D.D., Converso, A., Sharpless, K.B., Finn, M.G. 2,6-dichloro-9-thiabicyclo[3.3.1]nonane: multigram display of azides and cyanides components on a versatile scaffold. Molecules 11:212, 2006.

Dìaz, D.D., Finn, M.G., Mishima, M. Substituent effects on the gas-phase basicity of formamidine ureas. Eur. J. Org. Chem. 235, 2006, Issue 1.

Dìaz, D.D., Lewis, W.G., Finn, M.G. Activation of urea as a leaving group in substitution reactions of formamidine ureas. Chem. Lett. 34:78, 2005.

Dìaz, D.D., Rajagopal, K., Strable, E., Schneider, J., Finn, M.G. “Click” chemistry in a supramolecular environment: stabilization of organogels by copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition. J. Am. Chem. Soc. 128:6056, 2006.

Dìaz, D.D., Sen Gupta, S., Kuzelka, J., Cymborowski, M., Sabat, M., Finn, M.G. Bis(formamidine-urea) complexes of NiII and CuII: synthesis, characterization, and reactivity. Eur. J. Inorg. Chem. 4489, 2006, Issue 22.

Finn, M.G. Emerging high-throughput screening methods for asymmetric induction. In: Chiral Analysis. Busch, K.W., Busch, M.A. (Eds.). Elsevier, St. Louis, in press.

Johnson, J.A., Lewis, D.R., Dìaz, D.D., Finn, M.G., Koberstein, J.T., Turro, N.J. Synthesis of degradable model networks via ATRP and click chemistry. J. Am. Chem. Soc. 128:6564, 2006.

Li, C., Finn, M.G. Click chemistry in materials synthesis, 2: acid-swellable crosslinked polymers made by copper-catalyzed azide-alkyne cycloaddition. J. Polym. Sci. A Polym. Chem. 44:5513, 2006.

Punna, S., Kaltgrad, E., Finn, M.G. “Clickable” agarose for affinity chromatography. Bioconjug. Chem. 16:1536, 2005.

Sen Gupta, S., Kuzelka, J., Singh, P., Lewis, W.G., Manchester, M., Finn, M.G. Accelerated bioorthogonal conjugation: a practical method for the ligation of diverse functional molecules to a polyvalent virus scaffold. Bioconjug. Chem. 16:1572, 2005.

Whiting, M., Muldoon, J., Lin, Y.-C., Silverman, S.M., Lindstrom, W., Olson, A.J., Kolb, H.C., Finn, M.G., Sharpless, K.B., Elder, J.H., Fokin, V.V. Inhibitors of HIV-1 protease via in situ click chemistry. Angew. Chem. Int. Ed. 45:1435, 2006.

Wu, P., Malkoch, M., Hunt, J., Vestberg, R., Kaltgrad, E., Finn, M.G., Fokin, V.V., Sharpless, K.B., Hawker, C.J. Multivalent, bifunctional dendrimers prepared by click chemistry. Chem. Commun. (Camb.) 5775, 2005, Issue 46.

Yang, H., Das, N., Huang, F., Hawkridge, A.M., Arif, A.M., Finn, M.G., Muddiman, D.C., Stang, P.J. Incorporation of 2,6-di(4,4′-dipyridyl)-9-thiabicyclo[3.3.1]-nonane into discrete 2D supramolecules via coordination-driven self-assembly. J. Org. Chem. 71:6644, 2006.