A New Paradigm in Enantioselective Cobalt Catalysis: Cationic Cobalt(I) Catalysts for Heterodimerization, Cycloaddition, and Hydrofunctionalization Reactions of Olefins.

One of the major challenges facing organic synthesis in the 21st century is the utilization of abundantly available feedstock chemicals for fine chemical synthesis. Regio- and enantioselective union of easily accessible 1,3-dienes and other feedstocks like ethylene, alkyl acrylates, and aldehydes can provide valuable building blocks adorned with latent functionalities for further synthetic elaboration. Through an approach that relies on mechanistic insights and systematic examination of ligand and counterion effects, we developed an efficient cobalt-based catalytic system [(P∼P)CoX2/Me3Al] (P∼P = bisphosphine) to effect the first enantioselective heterodimerization of several types of 1,3-dienes with ethylene. In addition to simple cyclic and acyclic dienes, siloxy-1,3-dienes participate in this reaction, giving highly functionalized, nearly enantiopure silyl enolates, which can be used for subsequent C-C and C-X bond-forming reactions. As our understanding of the mechanism of this reaction improved, our attention was drawn to more challenging partners like alkyl acrylates (one of the largest volume feedstocks) as the olefin partners instead of ethylene. Prompted by the intrinsic limitations of using aluminum alkyls as the activators for this reaction, we explored the fundamental chemistry of the lesser known (P∼P)Co(I)X species and discovered that in the presence of halide sequestering agents, such as sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaBARF) or (C6F5)3B, certain chiral bisphosphine complexes are superb catalysts for regio- and enantioselective heterodimerization of 1,3-dienes and alkyl acrylates. We have since found that these cationic Co(I) catalysts, most conveniently prepared in situ by reduction of the corresponding cobalt(II) halide complexes by zinc in the presence of NaBARF, promote enantioselective [2 + 2]-cycloaddition between alkynes and an astonishing variety of alkenyl derivatives to give highly functionalized cyclobutenes. In reactions between 1,3-enynes and ethylene, the [2 + 2]-cycloaddition between the alkyne and ethylene is followed by a 1,4-addition of ethylene in a tandem fashion to give nearly enantiopure cyclobutanes with an all-carbon quaternary center, giving a set of molecules that maps well into many medicinally relevant compounds. In another application, we find that the cationic Co(I)-catalysts promote highly selective hydroacylation and 1,2-hydroboration of prochiral 1,3-dienes. Further, we find that a cationic Co(I)-catalyst promotes cycloisomerization followed by hydroalkenylation of 1,6-enynes to produce highly functionalized carbo- and heterocyclic compounds. Surprisingly the regioselectivity of the alkene addition depends on whether it is a simple alkene or an acrylate, and the acrylate addition produces an uncommon Z-adduct. This Account will provide a summary of the enabling basic discoveries and the attendant developments that led to the unique cationic Co(I)-complexes as catalysts for disparate C-C and C-B bond-forming reactions. It is our hope that this Account will stimulate further work with these highly versatile catalysts which are derived from an earth-abundant metal.

FRETing about the details: Case studies in the use of a genetically encoded fluorescent amino acid for distance-dependent energy transfer.

Förster resonance energy transfer (FRET) is a valuable method for monitoring protein conformation and biomolecular interactions. Intrinsically fluorescent amino acids that can be genetically encoded, such as acridonylalanine (Acd), are particularly useful for FRET studies. However, quantitative interpretation of FRET data to derive distance information requires careful use of controls and consideration of photophysical effects. Here we present two case studies illustrating how Acd can be used in FRET experiments to study small molecule induced conformational changes and multicomponent biomolecular complexes.

Identification of a Putative α-synuclein Radioligand Using an in silico Similarity Search.

PURPOSE: Previous studies from our lab utilized an ultra-high throughput screening method to identify compound 1 as a small molecule that binds to alpha-synuclein (α-synuclein) fibrils. The goal of the current study was to conduct a similarity search of 1 to identify structural analogs having improved in vitro binding properties for this target that could be labeled with radionuclides for both in vitro and in vivo studies for measuring α-synuclein aggregates. METHODS: Using 1 as a lead compound in a similarity search, isoxazole derivative 15 was identified to bind to α-synuclein fibrils with high affinity in competition binding assays. A photocrosslinkable version was used to confirm binding site preference. Derivative 21, the iodo-analog of 15, was synthesized, and subsequently radiolabeled isotopologs [125I]21 and [11C]21 were successfully synthesized for use in in vitro and in vivo studies, respectively. [125I]21 was used in radioligand binding studies in post-mortem Parkinson's disease (PD) and Alzheimer's disease (AD) brain homogenates. In vivo imaging of an α-synuclein mouse model and non-human primates was performed with [11C]21. RESULTS: In silico molecular docking and molecular dynamic simulation studies for a panel of compounds identified through a similarity search, were shown to correlate with Ki values obtained from in vitro binding studies. Improved affinity of isoxazole derivative 15 for α-synuclein binding site 9 was indicated by photocrosslinking studies with CLX10. Design and successful (radio)synthesis of iodo-analog 21 of isoxazole derivative 15 enabled further in vitro and in vivo evaluation. Kd values obtained in vitro with [125I]21 for α-synuclein and Aß42 fibrils were 0.48 ± 0.08 nM and 2.47 ± 1.30 nM, respectively. [125I]21 showed higher binding in human postmortem PD brain tissue compared with AD tissue, and low binding in control brain tissue. Lastly, in vivo preclinical PET imaging showed elevated retention of [11C]21 in PFF-injected mouse brain. However, in PBS-injected control mouse brain, slow washout of the tracer indicates high non-specific binding. [11C]21 showed high initial brain uptake in a healthy non-human primate, followed by fast washout that may be caused by rapid metabolic rate (21% intact [11C]21 in blood at 5 min p.i.). CONCLUSION: Through a relatively simple ligand-based similarity search, we identified a new radioligand that binds with high affinity (<10 nM) to α-synuclein fibrils and PD tissue. Although the radioligand has suboptimal selectivity for α-synuclein towards Aß and high non-specific binding, we show here that a simple in silico approach is a promising strategy to identify novel ligands for target proteins in the CNS with the potential to be radiolabeled for PET neuroimaging studies.

A Novel Brain PET Radiotracer for Imaging Alpha Synuclein Fibrils in Multiple System Atrophy.

Abnormal α-synuclein (α-syn) aggregation characterizes α-synucleinopathies, including Parkinson's disease (PD) and multiple system atrophy (MSA). However, no suitable positron emission tomography (PET) radiotracer for imaging α-syn in PD and MSA exists currently. Our structure-activity relationship studies identified 4-methoxy-N-(4-(3-(pyridin-2-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)phenyl)benzamide (4i) as a PET radiotracer candidate for imaging α-syn. In vitro assays revealed high binding of 4i to recombinant α-syn fibrils (inhibition constant (Ki) = 6.1 nM) and low affinity for amyloid beta (Aß) fibrils in Alzheimer's disease (AD) homogenates. However, [3H]4i also exhibited high specific binding to AD, progressive supranuclear palsy, and corticobasal degeneration tissues as well as PD and MSA tissues, suggesting notable affinity to tau. Nevertheless, the specific binding to pathologic α-syn aggregates in MSA post-mortem brain tissues was significantly higher than in PD tissues. This finding demonstrated the potential use of [11C]4i as a PET tracer for imaging α-syn in MSA patients. Nonhuman primate PET studies confirmed good brain uptake and rapid washout for [11C]4i.

Generating Efficient Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetases for Structurally Diverse Non-Canonical Amino Acids.

Genetic code expansion (GCE) technologies commonly use the pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs from Methanosarcina mazei (Mm) and Methanosarcina barkeri (Mb) for site-specific incorporation of non-canonical amino acids (ncAAs) into proteins. Recently a homologous PylRS/tRNAPyl pair from Candidatus Methanomethylophilus alvus Mx1201 (Ma) was developed that, lacking the N-terminal tRNA-recognition domain of most PylRSs, overcomes insolubility, instability, and proteolysis issues seen with Mb/Mm PylRSs. An open question is how to alter Ma PylRS specificity to encode specific ncAAs with high efficiency. Prior work focused on "transplanting" ncAA substrate specificity by reconstructing the same active site mutations found in functional Mm/Mb PylRSs in Ma PylRS. Here, we found that this strategy produced low-efficiency Ma PylRSs for encoding three structurally diverse ncAAs: acridonyl-alanine (Acd), 3-nitro-tyrosine, and m-methyl-tetrazinyl-phenylalanine (Tet3.0-Me). On the other hand, efficient Ma PylRS variants were generated by a conventional life/death selection process from a large library of active site mutants: for Acd encoding, one variant was highly functional in HEK293T cells at just 10 µM Acd; for nitroY encoding, two variants also encoded 3-chloro, 3-bromo-, and 3-iodo-tyrosine at high efficiency; and for Tet-3.0-Me, all variants were more functional at lower ncAA concentrations. All Ma PylRS variants identified through selection had at least two different active site residues when compared with their Mb PylRS counterparts. We conclude that Ma and Mm/Mb PylRSs are sufficiently different that "active site transplantation" yields suboptimal Ma GCE systems. This work establishes a paradigm for expanding the utility of the promising Ma PylRS/tRNAPyl GCE platform.