The Skaggs Institute
for Chemical Biology

Scientific Report 2005

Viruses as Molecular Building Blocks

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

Research supported by the Skaggs Institute during the past several years has allowed us to pioneer the development of chemical methods to selectively attach functional molecules to the surfaces of virus particles. Combined with the ability to alter viruses by genetic methods, the chemical methods give us the capability to create virions for a wide variety of purposes. This past year, we made strong advances in these fundamental capabilities, added 2 new virus particles to our repertoire, and obtained exciting preliminary results indicating the usefulness of derivatized viruses in vitro and in mouse models of disease.

Fundamental Conjugation Reactions of Virus Particles

The copper-catalyzed azide-alkyne cycloaddition reaction, introduced in 2002 for efficient attachment of components to cowpea mosaic virus (CPMV), continues to be improved and applied in many laboratories around the world. We completed our article on optimal bioconjugation conditions this past year, and we have been working toward solving the last remaining problem of air sensitivity for the most active catalyst.

Carbohydrates and Polymer-Virus Conjugates

We recently showed that carbohydrates displayed on viral coat proteins elicit a strong immune response. Using chicken eggs, we isolated large quantities of polyclonal IgG antibodies to carbohydrates; these molecules bind selectively to trisaccharides and tetrasaccharides that are selective markers for breast, ovarian, and colon cancers. Both the method of antibody generation and the antibodies themselves are useful. We will pursue these studies in collaboration with the Center for Functional Glycomics at Scripps Research in the coming year.

Virus-Protein Conjugates

We recently discovered that the azide-alkyne cycloaddition reaction is uniquely suited to the demanding job of covering a virus particle with a large functional protein with a stable covalent linkage. The 80-kD protein transferrin, an iron-transport protein that is strongly upregulated in cancer cells, was arrayed on the surface of CPMV (Fig. 1).

>Fig. 1. Synthesis of a polyvalent transferring particle based on CPMV. The transferrin-alkyne and CPMV-azide were prepared via separate acylation reactions and then combined under the influence of a copper(I) catalyst. The product particles are larger and have a punctate pattern in transmission electron microscopy (bottom right) compared with the CPMV precursor (bottom left), revealing the presence of the attached large transferrin protein.

The resulting particles have advantageous pharmacokinetics and strong binding to target cells in vitro. Experiments with mouse tumor models are in progress to explore the properties of the particles in vivo.

A New Method to Measure Biodistribution of Proteins

Our preparation of gadolinium-labeled viruses for magnetic resonance imaging led to the observation that the concentrations of such particles can be measured with exquisite accuracy and sensitivity via a spectroscopic method: inductively coupled plasma optical emission spectroscopy. Using this technique, we can follow the distribution of gadolinium-labeled particles in the blood and major organs of mice with great ease and without the use of hazardous radioactive labels or cumbersome fluorescence methods. Because stable complexes of gadolinium and other inert lanthanide elements can be readily attached to proteins in a variety of ways, we are exploring this method as a general pharmacokinetic tool.

New Chemically Modified Viruses

This past year, using hepatitis B coat protein and azidohomoalanine in place of methionine, we achieved the first incorporation of unnatural amino acids into a viruslike particle. This method will allow us to program with complete accuracy the placement of the azide group and then to affect the group with complete selectivity by using copper-catalyzed cycloaddition. Work is proceeding with both hepatitis B virus and the bacteriophage Qβ.

Publications

Dìaz, D.D., Finn, M.G. A facile synthesis of N,N′-bis[formamidine]ureas and symmetrical N,N.-disubstituted formamidines. Lett. Org. Chem. 2:398, 2005.

Dìaz, D.D., Lewis, W.G., Finn, M.G. Acid-mediated amine exchange of N,N-dimethylformamidines: preparation of electron-rich formamidines. Synlett 2214, 2005, Issue 14.

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., Ripka, A.S., Finn, M.G. 1-(tert-Butylimino-methyl)-1,3-dimethyl-urea hydrochloride. Org. Synth. 82:59, 2005.

Gissibl, A., Finn, M.G., Reiser, O. Cu(II)-aza(bisoxazoline)-catalyzed asymmetric benzoylations. Org. Lett. 7:2325, 2005.

Keller, K.A., Guo, J., Punna, S., Finn, M.G. A thermally-cleavable linker for solid-phase synthesis. Tetrahedron Lett. 46:1181, 2005.

Meng, J., Fokin, V.V., Finn, M.G. Kinetic resolution by copper-catalyzed azide-alkyne cycloaddition. Tetrahedron Lett. 46:4543, 2005.

Narayan, S., Muldoon, J., Finn, M.G., Fokin, V.V., Kolb, H.C., Sharpless, K.B. “On water”: unique reactivity of organic compounds in aqueous suspension. Angew. Chem. Int. Ed. 44:3275, 2005.

Punna, S., Kuzelka, J., Wang, Q., Finn, M.G. Head-to-tail peptide cyclodimerization by copper-catalyzed azide-alkyne cycloaddition. Angew. Chem. Int. Ed. 44:2215, 2005.

Rae, C.S., Wei Khor, I., Wang, Q., Destito, G., Gonzalez, M.J., Singh, P., Thomas, D.M., Estrada, M.N., Powell, E., Finn, M.G., Manchester, M. Systemic trafficking of plant virus nanoparticles in mice via the oral route. Virology 343:224, 2005.

Rodionov, V.O., Fokin, V.V., Finn, M.G. Mechanism of the ligand-free Cu(I)-catalyzed azide-alkyne cycloaddition reaction. Angew. Chem. Int. Ed. 44:2210, 2005.

Sen Gupta, S., Raja, K.S., Kaltgrad, E., Strable, E., Finn, M.G. Virus-glycopolymer conjugates by copper(I) catalysis of atom transfer radical polymerization and azide-alkyne cycloaddition. Chem. Commun. (Camb.) 4315, 2005, Issue 34.

Strable, E., Johnson, J.E., Finn, M.G . Natural nanochemical building blocks: icosahedral virus particles organized by attached oligonucleotides. Nano Lett. 4:1385, 2004.

Stray, S.J., Bourne, C.R., Punna, S., Lewis, W.G., Finn, M.G., Zlotnick, A. A heteroaryldihydropyrimidine activates and can misdirect hepatitis B virus capsid assembly. Proc. Natl. Acad. U. S. A. 102:8138, 2005.