The Skaggs Institute
for Chemical Biology
Click and Virus-Based Chemistry for Biological Discovery
M.G. Finn, A. Accurso, S. Brown, R. Jagasia, D. Prasuhn, A. Udit
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.
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,
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.