News and Publications
The Skaggs Institute for Chemical Biology
Scientific Report 1998-1999
Freezing Molecules in Space and Time: Assembly and Encapsulation
J. Rebek, Jr., M. Brody, R. Castellano, Y.-L. Cho, S. Craig, G. Haberhauer,
T. Haino, F. Hof, S. Körner, S. Ma, T. Martín, S. Mecozzi, C. Nuckolls,
B. O'Leary, J.-U. Peters, K. Pryor, D. Pupowicz, P. Rasmussen, A. Renslo, J.
Rivera-Ortiz, D. Rudkevich, J. Santamaria, C. Schalley, L. Somogyi, S. Starnes,
N. Svenstrup, F. Tucci, A. Wartini, P. Wash
When several copies of a molecule interact and assemble, new behaviors emerge
that are not apparent in the individual molecules but are unique to their assemblies.
Predicting these behaviors is often as difficult as predicting the shape of a
honeycomb by studying a single bee. Our research focuses on these multimolecular
assemblies: how to make them, what forces hold them together, how fast they come
together, what sequence of events leads to them, and what are the assemblies
good for. These assemblies are dynamic, constantly forming and dissipating on
timescales that range from milliseconds to hours, long enough for many types
of molecular encounters and even chemical reactions to take place within the
assemblies. Getting good pictures of these assemblies is like hitting a moving
The assemblies we study have another feature: they form hollow, capsulelike
structures in which smaller molecules are temporarily captured. The result is
a single molecule that is temporarily isolated in space and time, completely
surrounded by the capsule. This arrangement allows us to measure the behavior
of individual molecules or single-molecule events. The capsules can be used as
sensors, as reaction chambers, and as vehicles for delivery across membranes.
Synthesis of the capsular assemblies is constantly improving. The latest methods
are inspired by biology in which a single structure can be used repeatedly in
different settings to give a diverse set of structures. In pursuing this modular
approach, we identified a glycoluril unit as this multipurpose module (Fig. 1).
The unit has a wealth of hydrogen-bonding sites, and the hydrogen bonds hold
the transitory assembly together. Because of its rigid skeleton, the module maintains
a single, predictable shape. This shape has a gentle curvature similar to a part
(roughly a sixth) of an eggshell.
We connect the various molecular scaffolds that position the modules for assembly
and by piecing these scaffolds together, form the spherical shell of a capsule.
In the capsule shown in Figure 1, a large cavity is created when the molecules
assemble. The volume of the cavity is several hundred cubic angstroms, and large
holes in the structure allow the rapid entrance and exit of small molecules.
Large molecules enter and exit slowly, and we can observe them inside by using
nuclear magnetic resonance spectroscopy. Overall, the capsules sort molecules
according to size; that is, the capsules act as "molecular sieves."
In a second type of synthesis, the capsule is formed by folding a single very
large molecule into the appropriate shape (Fig. 2). In this case, a bowl-shaped
molecule, a calixarene, was used to provide molecular curvature, and 2 of these
subunits were permanently linked. By coupling the 2 molecules, we reduced their
independence and their entropies and improved their chances of folding properly.
The capsule is formed when the 2 bowls come together, rim to rim. Urea groups
arranged along the rim provided the hydrogen bonds in this example. Assembly
of the capsule was enhanced, and the expected improvements in trapping small
molecules inside were detected. However, some unexpected features emerged: certain
of these molecules were able to form liquid crystals. Because of their ability
to encapsulate small molecules in this physical state, the assembles may have
unique applications as "smart" materials.
How much information or instruction is required to bring, for example, 4 individual
molecules together into a capsule? This question is a different way of looking
at entropy. We found that ureas and sulfonamides prefer hydrogen bonding to each
other rather than to themselves, and we used this information as a form of instruction
for assembly. A molecule was prepared with a urea at one end and a sulfonamide
at the other (Fig. 3). As before, the curvature of the molecule, corresponding
to a quarter of a sphere, was built into the glycoluril groups. The result was
a predictable arrangement of 4 subunits, head to tail. Even so, the capsule emerged
from its pieces only when the proper occupant was present to be trapped inside.
These occupants fill about 55% of the space in the capsule. We discovered that
this level of filling space is a powerful and perhaps universal driving force
for molecular recognition and assembly.
In another study, we are exploring the synthesis and properties of "open-ended" molecular
containers. We are preparing these containers with ever deepening cavities, and
we found that the molecule shown in Figure 4 can temporarily trap smaller fullerenes
such as C60. We are beginning to find practical applications for these
containers in molecular separations. Basic questions of how molecules get in
and out of the container have been answered: the 4 sides must fold down before
the resident occupant is displaced by the incoming one.
Molecular Diversity and Combinatorial Chemistry
We have synthesized structures in which a diversity of functional groups--acids
and bases, hydrophobic and hydrophilic surfaces, large and small groups, and
groups of all shapes--protrude from a single face of a molecular platform. One
example of such a platform is the triphenylene structure shown in Figure 5. The
ability of the platform to interact with small molecules comes from its 3-fold
symmetry and rigidity. The triphenylene forms complexes with flat structures
that just fit within it and have complementary chemical surfaces. Caffeine and
nitrated aromatic molecules are complexed.
The ability of the triphenylene
to interact with large molecular surfaces such as proteins is due to the large
surface area spanned by the groups indicated as the spheres in Figure 5. We place
these groups on the platform randomly and then screen them for a specific behavior.
These new platforms represent uncharted areas in the landscape of molecular diversity.
Brody, M.S., Schalley, C.A., Rudkevich, D.M., Rebek, J., Jr. Synthesis
and characterization of an intramolecularly self-assembled capsule. Angew. Chem.
Int. Ed., in press.
Martín, T., Obst, U., Rebek, J., Jr. Hydrogen-bonding preferences
and the filling of space provide information for molecular assembly and encapsulation.
Science 281:1842, 1998.
Mink, D., Mecozzi, S., Rebek, J., Jr. Natural products analogs as scaffolds
for supramolecular and combinatorial chemistry. Tetrahedron Lett. 39:5709, 1998.
Pryor, K.E., Rebek, J., Jr. Multifunctionalized glycolurils. Org. Lett.
Schalley, C.A., Martín, T., Obst, U., Rebek, J., Jr. Characterization
of encapsulation complexes in the gas phase and solution. J. Am. Chem. Soc. 121:2133,
Schalley, C.A., Rivera, J.M., Martín, T., Santamaria, J., Siuzdak,
G., Rebek, J., Jr. Structural examination of supramolecular architectures
by electrospray ionization mass spectrometry. Eur. J. Org. Chem. 6:1325, 1999.
Szabo, T., O'Leary, B., Rebek, J., Jr. Self-assembling sieves. Angew.
Chem. Int. Ed. 37:3410, 1998.
Tucci, F.C., Rudkevich, D.M., Rebek, J., Jr. Deeper cavitands. J. Org.
Chem. 64:4555, 1999.
Waldvogel, S.R., Wartini, A.R., Rasmussen, P.H., Rebek, J., Jr. A triphenylene
scaffold with C3v-symmetry and nanoscale dimensions. Tetrahedron Lett.
Evolution of Novel Enzymes
F.E. Romesberg, A.K. Ogawa, R. Jimenez, D.L. McMinn, Y. Wu, S. Roy, T.
Sera, G. Xia, D. Hannay, E. Meggers, L. Chen, M. Berger, O. Abou-Zied, J. Chin,
The focus of our laboratory is the development of novel methods to understand
protein function and evolution. We are interested in developing new approaches
for the study of function (protein dynamics) and new methods for expanding these
functions to include new activities (in vitro directed evolution).
EVOLUTION OF NOVEL CATALYTIC FUNCTION
Information storage and replication play a central role in life. The "genetic
alphabet" is composed of the letters G, C, A, and T, which designate the bases
guanine, cytosine, adenine, and thymine, respectively. These letters (or bases)
combine to form 2 base pairs as a result of specific patterns of hydrogen bonding.
We are interested in expanding the genetic alphabet by developing a third base
pair composed of unnatural nucleobases. We have been exploring hydrophobicity,
as opposed to hydrogen bonding, as a driving force for base pairing.
We are developing phage display methods, as well as general high-throughput
screening methods, for the in vitro evolution of DNA polymerases capable of efficient
and high-fidelity synthesis of DNA that contains unnatural nucleobases. We are
also developing phage display methods to evolve proteins with interesting dynamic
properties. For example, the enzyme photolyase harvests the energy of visible
light to catalyze repair of thymine dimers in DNA. We are developing methods
to evolve photolyase proteins with novel properties, such as increased rates
of energy transfer and electron transfer.
PROTEIN FUNCTION AND DYNAMICS
To understand protein dynamics, we are exploring the role of atomic motion,
in addition to domain motion and conformational changes, in protein function
and catalysis. Selective deuteration of proteins, at nonexchangeable positions,
results in a carbon-deuterium oscillator that absorbs light in an otherwise transparent
region of the infrared spectrum. This modification allows the selective deposition
or measurement of energy in a specific and localized protein vibration. Time-resolved
spectroscopy can then be used to determine the role of vibrational energy in
protein function and catalysis.
The first step in either natural or in vitro evolution is the generation of
a pool of diversity, which must be sufficient to drive evolution. We are interested
in using photon echo spectroscopy to characterize the diversity of protein libraries.
This technique allows the quantitative determination of the static inhomogeneity
that contributes to an electronic or vibrational linewidth. This inhomogeneity
may be related to functional binding-site diversity. We are characterizing natural
immunologic libraries of antibodies and in vitro libraries of protein with bound