22 research outputs found
Fragmentable Polycationic Materials Based on Anchimeric Assistance
A new
family of modular, fragmentable oligo- and polycations has
been developed based on the reactions of 9-thiabicyclo[3.3.1]Âdichloride
and related compounds with substituted dipyridyl nucleophiles by an
anchimeric assistance mechanism. Each bond-forming event in this condensation
polymerization process generates a positive charge in the main chain.
Product lengths were found to be dependent on the reactivity of the
electrophile, which was tunable by changing the nature of the leaving
group β to sulfur. The monomers were easily synthesized, and
the resulting readily available polymers were found to be highly efficient
binders of nucleic acid. They exhibited properties of cytotoxicity
and DNA transfection expected of such polycationic materials, but
with interesting structure–activity differences that remain
to be explored. The polycations decomposed by hydrolysis at rates
dependent on the leaving group ability of the pyridyl unit, which
correlated roughly with the p<i>K</i><sub>a</sub> of its
conjugate acid. Polymer decomposition occurs simultaneously throughout
the length of the chains, rather than from the ends; the decomposition
products were tested and found to be only minimally toxic to cultured
cells
Thiabicyclononane-Based Antimicrobial Polycations
BicycloÂ[3.3.1]Ânonane
(BCN) polycations were synthesized by the
reaction of the bivalent electrophile thiabicyclo[3.3.1]Ânonane dinitrate
with a series of simple bisÂ(pyridine) nucleophiles. Oligomers of moderate
chain length were formed in a modular approach that tolerated the
inclusion of functionalized and variable-length linkers between the
pyridine units. Post-polymerization modification via copper-catalyzed
azide–alkyne cyloaddition was enabled by the inclusion of terminal
alkyne groups in these monomers. Most of the resulting polymers, new
members of the polyionene class, inhibited the growth of bacteria
at the ÎĽg/mL level and killed static bacterial cells at polymer
concentrations of tens of ng/mL, with moderate to good selectivity
with respect to lysis of red blood cells. While resistance to the
BCN polymers was developed only very slowly over multiple passages,
a degradable version of the polycation was observed to make <i>E. coli</i> cells more susceptible to other quaternary ammonium
based antimicrobials. Solid substrates (glass and crystalline silicon)
covalently functionalized with a representative BCN polycation were
also able to repetitively kill bacteria in solution at high rates
and with cleaning by simple sonication between exposures
Tyrosine Cross-Linking Reveals Interfacial Dynamics in Adeno-Associated Viral Capsids during Infection
Viral capsid dynamics are often observed during infectious
events
such as cell surface attachment, entry and genome release. Structural
analysis of adeno-associated virus (AAV), a helper-dependent parvovirus,
revealed a cluster of surface-exposed tyrosine residues at the icosahedral
two-fold symmetry axis. We exploited the latter observation to carry
out selective oxidation of Tyr residues, which yielded cross-linked
viral protein (VP) subunit dimers, effectively “stitching”
together the AAV capsid two-fold interface. Characterization of different
Tyr-to-Phe mutants confirmed that the formation of cross-linked VP
dimers is mediated by dityrosine adducts and requires the Tyr704 residue,
which crosses over from one neighboring VP subunit to the other. When
compared to unmodified capsids, Tyr-cross-linked AAV displayed decreased
transduction efficiency in cell culture. Surprisingly, further biochemical
and quantitative microscopy studies revealed that restraining the
two-fold interface hinders externalization of buried VP N-termini,
which contain a phospholipase A2 domain and nuclear localization sequences
critical for infection. These adverse effects caused by tyrosine oxidation
support the notion that interfacial dynamics at the AAV capsid two-fold
symmetry axis play a role in externalization of VP N-termini during
infection
Traceless Release of Alcohols Using Thiol-Sensitive Oxanorbornadiene Linkers
A class of ester–amide
oxanorbornadiene (EA-OND) molecules
was developed to release alcohol cargos by succinimide formation upon
addition of a thiol reagent. The resulting ring-closed adducts undergo
further fragmentation by retro-Diels–Alder reaction to release
a furan moiety in a manner similar to oxanorbornadiene diesters. The
rates of each of these fragmentation pathways in the same medium were
found to be sensitive to the steric nature of the amide substituent.
Alcohol release was much faster in protic solvents than in aprotic
ones, suggesting that this system may be useful for rapid response
to thiols in biological environments. Accordingly, the attachment
and thiol-dependent release of cholesterol was characterized as an
example of the manipulation of a drug-like cargo
Selection of Natural Peptide Ligands for Copper-Catalyzed Azide–Alkyne Cycloaddition Catalysis
The
copper-catalyzed azide–alkyne cycloaddition (CuAAC)
reaction is a powerful tool for making connections in both organic
reactions and biological systems. However, the use of this ligation
process in living cells is limited by the toxicity associated with
unbound copper ions. As an initial attempt to create peptide-based
accelerating ligands capable of cellular expression, we performed
synthesis and selection for such species on solid-phase synthesis
beads bearing both candidate ligand and alkyne substrate. A simple
histidine-containing motif (HXXH) was identified, and found after
solution-phase optimization to produce single-turnover systems showing
moderate rate acceleration over the ligand-free reaction. CuAAC reaction
rates and yields for different alkynes were found to respond to the
peptide ligands, demonstrating a substrate scope beyond what was used
for the selection steps, but also illustrating the potential difficulty
in evolving a general CuAAC catalyst
Degradable Conjugates from Oxanorbornadiene Reagents
Oxanorbornadienedicarboxylate (OND) reagents were explored
for
purposes of binding and releasing drugs from serum albumins as representative
macromolecular carriers. Being highly reactive Michael acceptors,
ONDs form adducts with thiols and amines, which then undergo retro-Diels–Alder
fragmentation. A study of more than 30 model adducts revealed a number
of modifications that can be used to influence adduct stability. For
the most reactive OND linkers, the labeling of the single available
bovine serum albumin (BSA) cysteine residue was complete within minutes
at a mid-micromolar concentration of reactants. While a selectivity
of greater than 1000-fold for thiol over amine was observed with model
amino acids, the labeling of protein amines with ONDs is fast enough
to be practical, as demonstrated by the reaction with thiol-depleted
BSA. The OND–amine adducts were found to be up to 15 times
more stable than OND–thiol adducts, and to be sensitive to
acid by virtue of a stereochemically dependent acceleration of cycloreversion.
The release rate of fluorescent cargo from serum albumins was tuned
by selecting the coupling partners: the available half-lives ranged
from 40 min to 7 days at 37 °C. Such versatility of release profiles
from protein carriers, controlled by the nature of the OND linkage,
is a useful addition to the drug delivery toolbox
Relative Performance of Alkynes in Copper-Catalyzed Azide–Alkyne Cycloaddition
Copper-catalyzed azide–alkyne
cycloaddition (CuAAC) has
found numerous applications in a variety of fields. We report here
only modest differences in the reactivity of various classes of terminal
alkynes under typical bioconjugative and preparative organic conditions.
Propargyl compounds represent an excellent combination of azide reactivity,
ease of installation, and cost. Electronically activated propiolamides
are slightly more reactive, at the expense of increased propensity
for Michael addition. Certain alkynes, including tertiary propargyl
carbamates, are not suitable for bioconjugation due to copper-induced
fragmentation. A fluorogenic probe based on such reactivity is available
in one step from rhodamine 110 and can be useful for optimization
of CuAAC conditions
Direct Measurement of Trafficking of the Cystic Fibrosis Transmembrane Conductance Regulator to the Cell Surface and Binding to a Chemical Chaperone
Mutations
in the cystic fibrosis transmembrane conductance regulator
(CFTR) result in the disease cystic fibrosis. Deletion of Phe508,
the most prevalent mutation associated with this disease, disrupts
trafficking of the protein. Small molecule correctors yield moderate
improvements in the trafficking of ΔF508-CFTR to the plasma
membrane. It is currently not known if correctors increase the level
of trafficking through improved cargo loading of transport vesicles
or through direct binding to CFTR. Real-time measurements of trafficking
were utilized to identify the mechanistic details of chemical, biochemical,
and thermal factors that impact CFTR correction, using the corrector
molecule VX-809, a secondary mutation (I539T), and low-temperature
conditions. Each individually improved trafficking of ΔF508-CFTR
to approximately 10% of wild-type levels. The combination of VX-809
with either low temperature or the I539T mutation increased the amount
of CFTR on the plasma membrane to nearly 40%, indicating synergistic
activity. The number of vesicles reaching the surface was significantly
altered; however, the amount of channel in each vesicle remained the
same. Direct binding measurements of VX-809 in native membranes using
backscattering interferometry indicate tight binding to CFTR, which
occurred in a manner independent of mutation. The similar values obtained
for all forms of the channel indicate that the binding site is not
compromised or enhanced by these mutations
Glycan-Targeted Virus-like Nanoparticles for Photodynamic Therapy
Virus-like particles (VLPs) have proven to be versatile
platforms
for chemical and genetic functionalization for a variety of purposes
in biomedicine, catalysis, and materials science. We describe here
the simultaneous modification of the bacteriophage Qβ VLP with
a metalloporphyrin derivative for photodynamic therapy and a glycan
ligand for specific targeting of cells bearing the CD22 receptor.
This application benefits from the presence of the targeting function
and the delivery of a high local concentration of singlet oxygen-generating
payload
Membrane Association Dictates Ligand Specificity for the Innate Immune Receptor NOD2
The
human gut must regulate its immune response to resident and
pathogenic bacteria, numbering in the trillions. The peptidoglycan
component of the bacterial cell wall is a dense and rigid structure
that consists of polymeric carbohydrates and highly cross-linked peptides
which offers protection from the host and surrounding environment.
Nucleotide-binding oligomerization domain-containing protein 2 (NOD2),
a human membrane-associated innate immune receptor found in the gut
epithelium and mutated in an estimated 30% of Crohn’s disease
patients, binds to peptidoglycan fragments and initiates an immune
response. Using a combination of chemical synthesis, advanced analytical
assays, and protein biochemistry, we tested the binding of a variety
of synthetic peptidoglycan fragments to wild-type (WT)-NOD2. Only
when the protein was presented in the native membrane did binding
measurements correlate with a NOD2-dependent nuclear factor kappa-light-chain-enhancer
of activated B cells (NF-ÎşB) response, supporting the hypothesis
that the native-membrane environment confers ligand specificity to
the NOD2 receptor for NF-ÎşB signaling. While <i>N</i>-acetyl-muramyl dipeptide (MDP) has been thought to be the minimal
peptidoglycan fragment necessary to activate a NOD2-dependent immune
response, we found that fragments with and without the dipeptide moiety
are capable of binding <i>and</i> activating a NOD2-dependent
NF-ÎşB response, suggesting that the carbohydrate moiety of the
peptidoglycan fragments is the minimal functional epitope. This work
highlights the necessity of studying NOD2-ligand binding in systems
that resemble the receptor’s natural environment, as the cellular
membrane and/or NOD2 interacting partners appear to play a crucial
role in ligand binding and in triggering an innate immune response