Morphing low-affinity ligands into high-avidity nanoparticles by thermally triggered self-assembly of a genetically encoded polymer.

Multivalency is the increase in avidity resulting from the simultaneous interaction of multiple ligands with multiple receptors. This phenomenon, seen in antibody-antigen and virus-cell membrane interactions, is useful in designing bioinspired materials for targeted delivery of drugs or imaging agents. While increased avidity offered by multivalent targeting is attractive, it can also promote nonspecific receptor interaction in nontarget tissues, reducing the effectiveness of multivalent targeting. Here, we present a thermal targeting strategy--dynamic affinity modulation (DAM)--using elastin-like polypeptide diblock copolymers (ELP(BC)s) that self-assemble from a low-affinity to high-avidity state by a tunable thermal "switch", thereby restricting activity to the desired site of action. We used an in vitro cell binding assay to investigate the effect of the thermally triggered self-assembly of these ELP(BC)s on their receptor-mediated binding and cellular uptake. The data presented herein show that (1) ligand presentation does not disrupt ELP(BC) self-assembly; (2) both multivalent ligand presentation and upregulated receptor expression are needed for receptor-mediated interaction; (3) increased size of the hydrophobic segment of the block copolymer promotes multivalent interaction with membrane receptors, potentially due to changes in the nanoscale architecture of the micelle; and (4) nanoscale presentation of the ligand is important, as presentation of the ligand by micrometer-sized aggregates of an ELP showed a low level of binding/uptake by receptor-positive cells compared to its presentation on the corona of a micelle. These data validate the concept of thermally triggered DAM and provide rational design parameters for future applications of this technology for targeted drug delivery.

Development of Stimulus-Responsive Ligands for the Modulation of Copper and Iron Coordination

The ability to manipulate the coordination chemistry of metal ions has significant ramifications for the study and treatment of metal-related health concerns, including iron overload, UV skin damage, and microbial infection among many other conditions. To address this concern, chelating agents that change their metal binding characteristics in response to external stimuli have been synthesized and characterized by several spectroscopic and chromatographic analytical methods. The primary stimuli of interest for this work are light and hydrogen peroxide.

Herein we report the previously unrecognized photochemistry of aroylhydrazone metal chelator ((E)-N′-[1-(2-hydroxyphenyl)ethyliden]isonicotinoylhydrazide) (HAPI) and its relation to HAPI metal binding properties. Based on promising initial results, a series of HAPI analogues was prepared to probe the structure-function relationships of aroylhydrazone photochemistry. These efforts elucidate the tunable nature of several aroylhydrazone photoswitching properties.

Ongoing efforts in this laboratory seek to develop compounds called prochelators that exhibit a switch from low to high metal binding affinity upon activation by a stimulus of interest. In this context, we present new strategies to install multiple desired functions into a single structure. The prochelator 2-((E)-1-(2-isonicotinoylhydrazono)ethyl)phenyl (E)-3-(2,4-dihydroxyphenyl)acrylate (PC-HAPI) is masked with a photolabile trans-cinnamic acid protecting group that releases umbelliferone, a UV-absorbing, antioxidant coumarin along with a chelating agent upon UV irradiation. In addition to the antioxidant effects of the coumarin, the released chelator (HAPI) inhibits metal-catalyzed production of damaging reactive oxygen species. Finally a peroxide-sensitive prochelator quinolin-8-yl (Z)-3-(4-hydroxy-2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)phenyl)acrylate (BCQ) has been prepared using a novel synthetic route for functionalized cis-cinnamate esters. BCQ uses a novel masking strategy to trigger a 90-fold increase in fluorescence emission, along with the release of a desired chelator, in the presence of hydrogen peroxide.

The Synthesis of Novel N-Heterocyclic Scaffolds and Diazirine-Based Molecular Tags

N-Heterocycles are ubiquitous in biologically active natural products and pharmaceuticals. Yet, new syntheses and modifications of N-heterocycles are continually of interest for the purposes of expanding chemical space, finding quicker synthetic routes, better pharmaceuticals, and even new handles for molecular labeling. There are several iterations of molecular labeling; the decision of where to place the label is as important as of which visualization technique to emphasize.

Piperidine and indole are two of the most widely distributed N-heterocycles and thus were targeted for synthesis, functionalization, and labeling. The major functionalization of these scaffolds should include a nitrogen atom, while the inclusion of other groups will expand the utility of the method. Towards this goal, ease of synthesis and elimination of step-wise transformations are of the utmost concern. Here, the concept of electrophilic amination can be utilized as a way of introducing complex secondary and tertiary amines with minimal operations.

Molecular tags should be on or adjacent to an N-heterocycle as they are normally the motifs implicated at the binding site of enzymes and receptors. The labeling techniques should be useful to a chemical biologist, but should also in theory be useful to the medical community. The two types of labeling that are of interest to a chemist and a physician would be positron emission tomography (PET) and magnetic resonance imaging (MRI).

Coincidentally, the 3-positions of both piperidine and indole are historically difficult to access and modify. However, using electrophilic amination techniques, 3-functionalized piperidines can be synthesized in good yields from unsaturated amines. In the same manner, 3-labeled piperidines can be obtained; the piperidines can either be labeled with an azide for biochemical research or an 18F for PET imaging research. The novel electrophiles, N-benzenesulfonyloxyamides, can be reacted with indole in one of two ways: 3-amidation or 1-amidomethylation, depending on the exact reaction conditions. Lastly, a novel, hyperpolarizable 15N2-labeled diazirine has been developed as an exogenous and versatile tag for use in magnetic resonance imaging.