Synthesis of Differentially Protected Azatryptophan Analogs via Pd2(dba)3/XPhos Catalyzed Negishi Coupling of N-Ts Azaindole Halides with Zinc Derivative from Fmoc-Protected tert-Butyl (R)-2-Amino-3-iodopropanoate.

Unnatural amino acids play an important role in peptide based drug discovery. Herein, we report a class of differentially protected azatryptophan derivatives synthesized from N-tosyl-3-haloazaindoles 1 and Fmoc-protected tert-butyl iodoalanine 2 via a Negishi coupling. Through ligand screening, Pd2(dba)3/XPhos was found to be a superior catalyst for the coupling of 1 with the zinc derivative of 2 to give tert-butyl (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propanoate derivatives 3 in 69-91% isolated yields. In addition, we have demonstrated that the protecting groups, namely, Ts, Fmoc, and tBu, can be easily removed selectively.

Ligand-directed delivery of fluorophores to track native calcium-permeable AMPA receptors in neuronal cultures.

Subcellular trafficking of neuronal receptors is known to play a key role in synaptic development, homeostasis, and plasticity. We have developed a ligand-targeted and photo-cleavable probe for delivering a synthetic fluorophore to AMPA receptors natively expressed in neurons. After a receptor is bound to the ligand portion of the probe molecule, a proteinaceous nucleophile reacts with an electrophile on the probe, covalently bonding the two species. The ligand may then be removed by photolysis, returning the receptor to its non-liganded state while leaving intact the new covalent bond between the receptor and the fluorophore. This strategy was used to label polyamine-sensitive receptors, including calcium-permeable AMPA receptors, in live hippocampal neurons from rats. Here, we describe experiments where we examined specificity, competition, and concentration on labeling efficacy as well as quantified receptor trafficking. Pharmacological competition during the labeling step with either a competitive or non-competitive glutamate receptor antagonist prevented the majority of labeling observed without a blocker. In other experiments, labeled receptors were observed to alter their locations and we were able to track and quantify their movements. We used a small molecule, ligand-directed probe to deliver synthetic fluorophores to endogenously expressed glutamate receptors for the purpose of tracking these receptors on live, hippocampal neurons. We found that clusters of receptors appear to move at similar rates to previous studies. We also found that the polyamine toxin pharmacophore likely binds to receptors in addition to calcium-permeable AMPA receptors.

Silent, fluorescent labeling of native neuronal receptors.

We have developed a minimally-perturbing strategy that enables labeling and subcellular visualization of endogenous dendritic receptors on live, wild-type neurons. Specifically, calcium-permeable non-NMDA glutamate receptors expressed in hippocampal neurons can be targeted with this novel synthetic tri-functional molecule. This ligand-directed probe was targeted towards AMPA receptors and bears an electrophilic group for covalent bond formation with an amino acid side chain on the extracellular side of the ion channel. This molecule was designed in such a way that the use-dependent, polyamine-based ligand accumulates the chemically-reactive group at the extracellular side of these polyamine-sensitive receptors, thereby allowing covalent bond formation between an electrophilic moiety on the nanoprobe and a nucleophilic amino acid sidechain on the receptor. Bioconjugation of this molecule results in a stable covalent bond between the nanoprobe and the target receptor. Subsequent photolysis of a portion of the nanoprobe may then be employed to effect ligand release allowing the receptor to re-enter the non-liganded state, all the while retaining the fluorescent beacon for visualization. This technology allows for rapid fluorescent labeling of native polyamine-sensitive receptors and further advances the field of fluorescent labeling of native biological molecules.

The GABA(A) receptor as a target for photochromic molecules.

Photochromic ligands, molecules that can be induced to change their physical properties through applied light, are currently the topic of much chemical biology research. This specialized class of small organic structures are, surprisingly to many, fairly common in nature. At the core of a number of natural biological processes lies a small molecule that changes shape or some other measurable property in response to light absorption. For instance, conformational changes invoked by reversible photoisomerization of a retinoid small molecule found in the photoreceptors of the human eye leads to vision. In plants, photoisomerization of a cinnamate moiety leads to altered gene expression. The photosensitive molecule can be viewed simply as a nanosensor of light, much like a photosensitive electrical component might be added to a circuit to sense day versus night to turn an electrical circuit on or off. Synthetic organic chemists and chemical biologists have been, for at least the last 15years, trying to either mimic or exploit the native photochromism found in nature. Here, we describe the design process to develop a photochromic molecule to be used in neurobiology.

Engineering a light-regulated GABAA receptor for optical control of neural inhibition.

Optogenetics has become an emerging technique for neuroscience investigations owing to the great spatiotemporal precision and the target selectivity it provides. Here we extend the optogenetic strategy to GABAA receptors (GABAARs), the major mediators of inhibitory neurotransmission in the brain. We generated a light-regulated GABAA receptor (LiGABAR) by conjugating a photoswitchable tethered ligand (PTL) onto a mutant receptor containing the cysteine-substituted α1-subunit. The installed PTL can be advanced to or retracted from the GABA-binding pocket with 500 and 380 nm light, respectively, resulting in photoswitchable receptor antagonism. In hippocampal neurons, this LiGABAR enabled a robust photoregulation of inhibitory postsynaptic currents. Moreover, it allowed reversible photocontrol over neuron excitation in response to presynaptic stimulation. LiGABAR thus provides a powerful means for functional and mechanistic investigations of GABAAR-mediated neural inhibition.

Prodrug approaches to reduce hyperexcitation in the CNS.

Hyperexcitation in the central nervous system is the root cause of a number of disorders of the brain ranging from acute injury to chronic and progressive diseases. The major limitation to treatment of these ailments is the miniscule, yet formidable blood-brain barrier. To deliver therapeutic agents to the site of desired action, a number of biomedical engineering strategies have been developed including prodrug formulations that allow for either passive diffusion or active transport across this barrier. In the case of prodrugs, once in the brain compartment, the active therapeutic agent is released. In this review, we discuss in some detail a number of factors related to treatment of central nervous system hyperexcitation including molecular targets, disorders, prodrug strategies, and focused case studies of a number of therapeutics that are at a variety of stages of clinical development.