The Skaggs Institute
for Chemical Biology

Scientific Report 2007

Click and Virus-Based Chemistry for Biological Discovery

M.G. Finn, A. Accurso, S. Brown, R. Jagasia, D. Prasuhn, A. Udit

Self-assembled particles composed of viral coat proteins constitute nanoscale scaffolds and cages of unmatched stability, ease of production, and genetic tailorability. During the past several years, with crucial support from the Skaggs Institute, we have developed methods for the chemical derivatization of these structures, enabling us to bring the full power of both chemistry and molecular biology to bear in the creation of biologically active large structures. The chemical methods are derived from click chemistry, the field of highly reliable synthetic methods developed and popularized by scientists in the laboratories of K.B. Sharpless, the Skaggs Institute, V.V. Fokin, Scripps Research, and in our laboratory. We have focused primarily on the copper-catalyzed azide-alkyne cycloaddition reaction. Use of this process on virus scaffolds made possible the following advances in the past year.

Fundamental Conjugation Reactions of Virus Particles

An electrochemical version of the copper azide-alkyne reaction has been refined and is now the method of choice for demanding instances of bioconjugation. Several new ligands that accelerate the process have been prepared on a large scale and are being used in laboratories around the world.

We have also completed our initial exploration of introducing azide- and alkyne-containing unnatural amino acids into our virus scaffolds, with subsequent coupling to complementary reagents via the copper-catalyzed reaction. With this method, we can prepare proteins with any functional group desired, at almost any position in the polypeptide chain.

Polyvalent Carbohydrate Conjugates

We discovered this year that carbohydrates become highly antigenic when displayed on the surface of the viral protein capsids. This discovery is significant because many pathogens and disease states are marked by the display of unusual surface glycans. If immune responses can be induced against these surface glycans, creation of novel vaccines may be possible. Figure 1 shows an example of the binding specificity of our polyclonal antibody raised in chickens (and isolated in large quantities from eggs) against the carbohydrate globo-H, which is a cancer marker, and that of a commercial monoclonal antibody against the same sugar. The binding selectivities and potencies are quite similar for the 2 antibodies, but the polyclonal material is obtained at approximately one thousandth of the cost of the monoclonal. antibody.

Fig. 1. Analysis of binding of polyclonal and monoclonal antibodies to globo-H to a wide collection of carbohydrates. The vertical lines mark the relative binding affinity to each of 264 sugars printed on an array. The colored shapes represent a code denoting the structures of each of the glycans to which a strong response was noted.

To take advantage of the antigenicity imparted by virus display, we have attached structures rich in mannose to the surface of our particles to mimic the presentation of these glycans by HIV. The resulting structures bind tightly to the HIV neutralizing antibody 2G12 discovered by D.R. Burton and coworkers, Scripps Research, and elicit a strong antiglycan immune response in test animals. These results are sufficiently promising to justify the continued refinement of the approach, and we are constructing new particles for this purpose. Click chemistry methods are essential to achieve the tight packing of glycans on the viral surface required for anti-HIV activity.

Synthesis of Cyclic Peptides

Support of our click chemistry efforts by the Skaggs Institute has also allowed us to investigate the unusual finding shown in Figure 2. When used to link azides and alkynes on resin-bound synthetic peptides, the copper-catalyzed reaction mediates an unprecedented cyclic dimerization reaction. Rings as large as 36 amino acids have been made selectively and in high yield. We have determined that this process is driven by the bimetallic nature of the reaction and the ability of peptide chains to orient themselves in a head-to-tail fashion by hydrogen bonding. Cyclic peptides have important uses as analogs to natural product–based antimicrobial agents and enzyme inhibitors.

Fig. 2. Cyclodimerization of peptides by copper-catalyzed azide-alkyne cycloaddition.

Publications

Bourne, C.R., Finn, M.G., Zlotnick A. Global structural changes in hepatitis B virus capsids induced by the assembly effector HAP1. J. Virol. 80:11055, 2006.

Destito, G., Yeh, R., Rae, C.S., Finn, M.G., Manchester, M. Folic acid-mediated targeting of cowpea mosaic virus particles to tumor cells. Chem. Biol. 14:1152, 2007.

Kaltgrad, E., Sen Gupta, S., Punna, S., Huang, C.-Y., Chang, A., Wong, C.-H., Finn, M.G., Blixt, O. Anti-carbohydrate antibodies elicited by polyvalent display on a viral scaffold. Chembiochem 8:1455, 2007.

Prasuhn, D.E., Jr., Yeh, R.M., Obenaus, A., Manchester, M., Finn, M.G. Viral MRI contrast agents: coordination of Gd by native virions and attachment of Gd complexes by azide-alkyne cycloaddition. Chem. Commun. (Camb.) 1269, 2007, Issue 12.

Rodionov, V.O., Presolski, S., Dìaz, D.D., Fokin, V.V., Finn, M.G. Ligand-accelerated Cu-catalyzed azide-alkyne cycloaddition: a mechanistic report. J. Am. Chem. Soc. 129:12705, 2007.

Rodionov, V.O., Presolski, S., Gardinier, S., Lim, Y.-H., Finn, M.G. Benzimidazole and related ligands for Cu-catalyzed azide-alkyne cycloaddition. J. Am. Chem. Soc. 129:12696, 2007.

Singh, P., Prasuhn, D., Yeh, R.M., Destito, G., Rae, C.S., Osborn, K., Finn, M.G., Manchester, M. Bio-distribution, toxicity, and pathology of cowpea mosaic virus nanoparticles in vivo. J. Control. Release 120:41, 2007.