At
the core of our efforts is an appreciation for the mechanisms of chemical reactivity
and the power of molecular biology to create useful tools for chemists,
chemical biologists, and materials scientists. Below you will find brief
descriptions of our current projects and links to representative papers
in those areas.
COMBINING
MOLECULAR BIOLOGY with CHEMISTRY: VIRUSES as MOLECULAR
BUILDING BLOCKS
About 10
years ago, we helped pioneer the use of organic
chemistry techniques to modify virus particles, and we have parlayed
those capabilities into a wide variety of applications. Our
most commonly used particles are currently the capsid
and . The
former is expressed in large quantities in E. coli cells as
a non-infectious virus-like particle, whereas the latter is grown in
pea plants. Both particles are highly stable toward extremes of pH,
temperature, solvent composition, and reagents, allowing them to be
modified by a variety of chemical reactions.
Virus particles
are many times larger than almost all products of synthetic chemistry,
and many times smaller than most cells. But they are
not so large as to be out of reach
of chemical techniques,
and they are large enough to present surfaces of biologically relevant
size to cells and tissues. Combining organic chemistry and molecular
biology to create and modify
these particles is extraordinarily powerful.
Viruses
are also highly regular structures, presenting multiple copies of
identical functionality on their external sand internal surfaces.
Dendrimers, polymers, metal nanoparticles, and cells all have
some of these qualities, but viruses are unique: no
other entities
of comparable dimensions are available with structures known and
controllable to atomic resolution. Such well-defined polyvalency
is crucial to the
biological function of viruses and of critical
importance to our efforts to use them in
many different applications.
Shown here are CPMV particles with inserted cysteine
residues (red
dots) providing attachment points in different patterns.
At
the present time, research is ongoing in the following areas:
Chemical
Fundamentals: New
methods of ,
including the use of enhancing
particle stability; learning the chemistry of new viruses particles.
Polyvalent
Presentation to Biology: Attachment
of biologically active compounds, from
to and
exploration of their .
This work includes the display of ,
peptides, and drug molecules, and with
cellular receptors and tissues. At the right is a representation of
a cryo-EM reconstruction of transferrin molecules attached to the
surface of the Q particle,
making a nanostructure that is taken up by cancer cells with remarkable
efficiency. Especially important in current projects is the display
of on
the surfaces of virus-like particles, in order to bring new functions
and attachment points to these scaffolds.
Immunology: Virus
particles are naturally immunogenic and have been used for many years
as antigens or carriers for antigenic molecules. To this field we have
added the idea that benefit
in similar ways from being displayed on ,
and we are exploring a variety of systems with immunology collaborators.
Our emphasis is on anti-cancer and anti-bacterial vaccine development.
Catalysis: We
have developed a practical method for the preparation of Q virus-like
particles .
When packaged in this way, the enzymes are highly active when their
substrates and products can diffuse through the capsid shell, and are
protected from denaturating and hydrolysis by proteases. This technology,
represented in cartoon form at the right, is quite general, which creates
opportunities for interesting reaction cascades and therapeutic applications.
Nanoparticle
Assemblies and Materials Science:
The
marriage of biological nanoparticles with aqueous-compatible methods
of polymer synthesis allows us to prepare highly monodisperse particles
with .
One can also attach moieties to virus particles that mediate their
into
higher-order structures. Our goals in include applications
to immunological shielding, light harvesting, catalysis, and molecular
sensing.
Selection
and Evolution: Creation
of virus libraries selection for function, retaining the property
of high expression yields.
Our
collaborators,
with whom we greatly enjoy working, include: (UCSD) – virus
targeting to cells and tissues (Scripps) – anti-HIV
vaccine development (Michigan
State) and (UC
Irvine)
– vaccines
against tumor-associated carbohydrate antigens
(Technische
Universität Munich) – cell
targeting with integrin-binding ligands (Scripps) – polyvalent
probes of immune cell binding (Scripps) – anti-bacterial
immune response, vaccine development (Scripps)
SYNTHETIC
METHODS AND CLICK CHEMISTRY
by
Kolb, Finn, and Sharpless, click chemistry involves the use of only the
most reliable, general, high-yielding, and byproduct-free organic reactions
for the construction of compounds with desired function. Its central
hypothesis is that most, if not all, chemical functions can be attained
by many different compounds among the nearly limitless possibilities
of three-dimensional structural space. If you restrict yourself as much
as possible to using only the best reactions, more diversity will be
accessible, since your reactions work with a wider variety of pieces.
One stands a better chance, therefore, of finding functional molecules
this way, although they will not look like the compounds made by nature
for the same purpose. To appreciate the possibilities, synthetic chemists
might ask themselves “what if every bond connection worked flawlessly,
regardless of the structure of the connecting pieces?” Others,
non-practitioners of the synthetic arts, might wonder what would be possible
if molecules of any desired shape or pattern of functional group display
were available without much trouble? We are not yet close to such ideals,
but the concept is powerful and enabling.
Click reactions usually use high-energy (“spring-loaded”)
reagents with well-defined reaction pathways, giving rise to selective
bond-forming events of wide scope. Examples include the nucleophilic
trapping of strained-ring electrophiles (epoxide, aziridines, aziridinium
ions, episulfonium ions), certain limited forms of carbonyl reactivity
(aldehydes + hydrazines or hydroxylamines, for example), and several
types of cycloaddition reactions. Our efforts using click chemistry include
three basic processes.
Cu-catalyzed
Azide-Alkyne Cycloaddition: This process,by
Valery Fokin, Barry Sharpless, and
colleagues for solution phase reactions (and by Morten Meldal and colleagues
for ),
has contributed dramatically to applications in materials
science, chemical biology, medicinal chemistry, and many other fields.
We have studied its mechanism and developed catalysts for use in demanding
cases of .
New improvements continue to
emerge as we use the reaction
with virus particles and
other.
Versatile
Electrophiles for Bioconjugation and Release: The
7-oxanorbornadienes derived from Diels-Alder cycloaddition
of furans and electron-deficient alkynes are an easily synthesized class
of compounds that serve as highly reactive electrophiles and interesting
cleavable linkages.
In our,we
showed them to be about as reactive as maleimides toward conjugate addition
of thiols,
yet more stable toward deactivation in aqueous solution and
able to undergo triggered retro-Diels-Alder cleavage. We are continuing
to develop this system and to explore its use in drug
delivery and materials science.
Reversible
Organic Linkages based on Anchimeric Assistance: Another
highly reliable reaction pattern is the substitution
reaction triggered by internal nucleophiles giving rise to high-energy
cyclic intermediates. An old example of this type of anchimeric assistance
with particularly
favorable properties wasby
us
and the Sharpless laboratory several years ago. Our group is carrying
this forward by exploring the of
the method and applying it to the synthesis of functional polymers and
surfaces.
MEDICINAL
CHEMISTRY
Enzyme
Inhibitors: Using techniques of ,
organic synthesis, and ,
we are developing small molecule agents against a panel of important
medicinal targets. These include thefor
control of nicotine addiction, drug transport enzymes for malaria treatment, for
AIDS treatment, for
autoimmune diseases, and .
In addition, we have several projects ongoing with collaborators at Pfizer.
These programs provide excellent training for group members in organic
synthesis, experience with the principles and applications
of medicinal
chemistry, and contacts with outstanding laboratories in both industry
and academia. The latter include Professors , , ,
,
,
and at
Scripps, Professor at
UCSD, and Professor and
colleagues at Scripps Florida.
MATERIALS
SCIENCE
The
branch of chemistry most heavily dependent on reactions which meet the
click chemistry standard is polymer synthesis. Indeed, the reliability
of polymerization reactions and the rich
functions of the products were
among the original inspirations for the click chemistry concept.
The identification of each new click reaction immediately enables the synthesis
of novel materials. With each of the synthetic methods described above,
we are making new and functional polymeric materials with interesting
applications.
These include (shown
here are graduate students
Adrian Accurso and Vu Hong demonstrating the
strength of one of Adrian’s adhesive
formulations), hydrogels for slow release of drug molecules, and biodegradable
materials for drug delivery.
ANALYTICAL
METHODS
The binding
of two molecules in solution invariably changes the structure, conformation,
and solvation states of the solutes. These factors contribute to the
overall refractive index of the solution, and therefore binding events
always change refractive index. Some years ago, of
Vanderbilt University realized this fact and built an instrument capable
of detecting changes in refractive index with the necessary sensitivity.
The technique, known as backscattering interferometry (BSI), requires
a simple laser-based optical train and is done at room temperature using
small amounts of analytes in a microfluidic channel. We collaborate with
the Bornhop group to further develop and apply this technique to biomolecular
interactions such as binding,
complexation
and the binding of to
their ligands or inhibitors. BSI is a label-free technique that works
with all sizes of molecules under a wide variety of conditions, including
serum, cell lysates, and crude membrane preparations. In conjunction
with the Bornhop group, we have built a BSI instrument in our laboratory
at Scripps. Research is ongoing into the fundamental aspects of the BSI
technique as well as in applications to many of the projects described
above.
capsid
and 
. The
former is expressed in large quantities in E. coli cells as
a non-infectious virus-like particle, whereas the latter is grown in
pea plants. Both particles are highly stable toward extremes of pH,
temperature, solvent composition, and reagents, allowing them to be
modified by a variety of chemical reactions. 
,
including the use of 
enhancing
particle stability; learning the chemistry of new viruses particles.
and
exploration of their 
.
This work includes the display of 
,
peptides, and drug molecules, and 
with
cellular receptors and tissues. At the right is a representation of
a cryo-EM reconstruction of transferrin molecules attached to the
surface of the Q
particle,
making a nanostructure that is taken up by cancer cells with remarkable
efficiency. Especially important in current projects is the display
of 
on
the surfaces of virus-like particles, in order to bring new functions
and attachment points to these scaffolds.
benefit
in similar ways from being displayed on 
,
and we are exploring a variety of systems with immunology collaborators.
Our emphasis is on anti-cancer and anti-bacterial vaccine development.
Catalysis: We
have developed a practical method for the preparation of Q
.
When packaged in this way, the enzymes are highly active when their
substrates and products can diffuse through the capsid shell, and are
protected from denaturating and hydrolysis by proteases. This technology,
represented in cartoon form at the right, is quite general, which creates
opportunities for interesting reaction cascades and therapeutic applications. 
particles
with 
.
One can also attach moieties to virus particles that mediate their
into
higher-order structures. Our goals in include applications
to immunological shielding, light harvesting, catalysis, and molecular
sensing.
(UCSD) – virus
targeting to cells and tissues 
(Scripps) – anti-HIV
vaccine development 
(Michigan
State) and 
(UC
Irvine)
(Technische
Universität Munich) – cell
targeting with integrin-binding ligands
(Scripps) – polyvalent
probes of immune cell binding 
(Scripps) – anti-bacterial
immune response, vaccine development 
(Scripps) 
by
Kolb, Finn, and Sharpless, click chemistry involves the use of only the
most reliable, general, high-yielding, and byproduct-free organic reactions
for the construction of compounds with desired function. Its central
hypothesis is that most, if not all, chemical functions can be attained
by many different compounds among the nearly limitless possibilities
of three-dimensional structural space. If you restrict yourself as much
as possible to using only the best reactions, more diversity will be
accessible, since your reactions work with a wider variety of pieces.
One stands a better chance, therefore, of finding functional molecules
this way, although they will not look like the compounds made by nature
for the same purpose. To appreciate the possibilities, synthetic chemists
might ask themselves “what if every bond connection worked flawlessly,
regardless of the structure of the connecting pieces?” Others,
non-practitioners of the synthetic arts, might wonder what would be possible
if molecules of any desired shape or pattern of functional group display
were available without much trouble? We are not yet close to such ideals,
but the concept is powerful and enabling. 
by
Valery Fokin, Barry Sharpless, and
colleagues for solution phase reactions (and by Morten Meldal and colleagues
for 
),
has contributed dramatically to applications in 
materials
science, chemical biology, medicinal chemistry, and many other fields.
We have studied its mechanism and developed catalysts for use in demanding
cases of 
.
New improvements continue to
emerge as we use the reaction
with virus particles and
other
.
cycloaddition
of furans and electron-deficient alkynes are an easily synthesized class
of compounds that serve as highly reactive electrophiles and interesting
cleavable linkages. 
,we
showed them to be about as reactive as maleimides toward conjugate addition
of thiols, 
substitution
reaction triggered by internal nucleophiles giving rise to high-energy
cyclic intermediates. An old example of this type of anchimeric assistance
with particularly
favorable properties was
by
us
and the Sharpless laboratory several years ago. Our group is carrying
this forward by exploring the 
of
the method and applying it to the synthesis of functional polymers and
surfaces. 
,
organic synthesis, and 
,
we are developing small molecule agents against a panel of important
medicinal targets. These include the
for
control of nicotine addiction, drug transport enzymes for malaria treatment,
for
AIDS treatment,
for
autoimmune diseases, and 
.
In addition, we have several projects ongoing with collaborators at Pfizer.
These programs provide excellent training for group members in organic
synthesis, experience with the principles and applications
of medicinal
chemistry, and contacts with outstanding laboratories in both industry
and academia. The latter include Professors 
, 
, 
,
,
, 
and 
at
Scripps, Professor 
at
UCSD, and Professor 
and
colleagues at Scripps Florida.
The
branch of chemistry most heavily dependent on reactions which meet the
click chemistry standard is polymer synthesis. Indeed, the reliability
of polymerization reactions and the rich 
(shown
here are graduate students 
of
Vanderbilt University realized this fact and built an instrument capable
of detecting changes in refractive index with the necessary sensitivity.
The technique, known as backscattering interferometry (BSI), requires
a simple laser-based optical train and is done at room temperature using
small amounts of analytes in a microfluidic channel. We collaborate with
the Bornhop group to further develop and apply this technique to biomolecular
interactions such as 
binding,
complexation
and the binding of 
to
their ligands or inhibitors. BSI is a label-free technique that works
with all sizes of molecules under a wide variety of conditions, including
serum, cell lysates, and crude membrane preparations. In conjunction
with the Bornhop group, we have built a BSI instrument in our laboratory
at Scripps. Research is ongoing into the fundamental aspects of the BSI
technique as well as in applications to many of the projects described
above. 
