Deciphering complexity in Pd-catalyzed cross-couplings.

Understanding complex reaction systems is critical in chemistry. While synthetic methods for selective formation of products are sought after, oftentimes it is the full reaction signature, i.e., complete profile of products/side-products, that informs mechanistic rationale and accelerates discovery chemistry. Here, we report a methodology using high-throughput experimentation and multivariate data analysis to examine the full signature of one of the most complicated chemical reactions catalyzed by palladium known in the chemical literature. A model Pd-catalyzed reaction was selected involving functionalization of 2-bromo-N-phenylbenzamide and multiple bond activation pathways. Principal component analysis, correspondence analysis and heatmaps with hierarchical clustering reveal the factors contributing to the variance in product distributions and show associations between solvents and reaction products. Using robust data from experiments performed with eight solvents, for four different reaction times at five different temperatures, we correlate side-products to a major dominant N-phenyl phenanthridinone product, and many other side products.

α-Functionalisation of Cyclic Sulfides Enabled by Lithiation Trapping.

A general and straightforward procedure for the lithiation trapping of cyclic sulfides such as tetrahydrothiophene, tetrahydrothiopyran and a thiomorpholine is described. Trapping with a wide range of electrophiles is demonstrated, leading to more than 50 diverse α-substituted saturated sulfur heterocycles. The methodology provides access to a range of α-substituted cyclic sulfides that are not easily synthesised by the currently available methods.

Exploration of piperidine 3D fragment chemical space: synthesis and 3D shape analysis of fragments derived from 20 regio- and diastereoisomers of methyl substituted pipecolinates.

Fragment-based drug discovery is now widely adopted for lead generation in the pharmaceutical industry. However, fragment screening collections are often predominantly populated with flat, 2D molecules. Herein, we report the synthesis of piperidine-based 3D fragment building blocks - 20 regio- and diastereoisomers of methyl substituted pipecolinates using simple and general synthetic methods. cis-Piperidines, accessed through a pyridine hydrogenation were transformed into their trans-diastereoisomers using conformational control and unified reaction conditions. Additionally, diastereoselective lithiation/trapping was utilised to access trans-piperidines. Analysis of a virtual library of fragments derived from the 20 cis- and trans-disubstituted piperidines showed that it consisted of 3D molecules with suitable molecular properties to be used in fragment-based drug discovery programs.

Platinum dissolution and tissue response following long-term electrical stimulation at high charge densities.

Objective. Established guidelines for safe levels of electrical stimulation for neural prostheses are based on a limited range of the stimulus parameters used clinically. Recent studies have reported particulate platinum (Pt) associated with long-term clinical use of these devices, highlighting the need for more carefully defined safety limits. We previously reported no adverse effects of Pt corrosion products in the cochleae of guinea pigs following 4 weeks of electrical stimulation using charge densities far greater than the published safe limits for cochlear implants. The present study examines the histopathological effects of Pt within the cochlea following continuous stimulation at a charge density well above the defined safe limits for periods up to 6 months.Approach. Six cats were bilaterally implanted with Pt electrode arrays and unilaterally stimulated using charge balanced current pulses at a charge density of 267µC cm-2phase-1using a tripolar electrode configuration. Electrochemical measurements were made throughout the implant duration and evoked potentials recorded at the outset and on completion of the stimulation program. Cochleae were examined histologically for particulate Pt, tissue response, and auditory nerve survival; electrodes were examined for surface corrosion; and cochlea, brain, kidney, and liver tissue analysed for trace levels of Pt.Main results. Chronic stimulation resulted in both a significant increase in tissue response and particulate Pt within the tissue capsule surrounding the electrode array compared with implanted, unstimulated control cochleae. Importantly, there was no stimulus-induced loss of auditory neurons (ANs) or increase in evoked potential thresholds. Stimulated electrodes were significantly more corroded compared with unstimulated electrodes. Trace analysis revealed Pt in both stimulated and control cochleae although significantly greater levels were detected within stimulated cochleae. There was no evidence of Pt in brain or liver; however, trace levels of Pt were recorded in the kidneys of two animals. Finally, increased charge storage capacity and charge injection limit reflected the more extensive electrode corrosion associated with stimulated electrodes.Significance. Long-term electrical stimulation of Pt electrodes at a charge density well above existing safety limits and nearly an order of magnitude higher than levels used clinically, does not adversely affect the AN population or reduce neural function, despite a stimulus-induced tissue response and the accumulation of Pt corrosion product. The mechanism resulting in Pt within the unstimulated cochlea is unclear, while the level of Pt observed systemically following stimulation at these very high charge densities does not appear to be of clinical significance.

Light- and Manganese-Initiated Borylation of Aryl Diazonium Salts: Mechanistic Insight on the Ultrafast Time-Scale Revealed by Time-Resolved Spectroscopic Analysis.

Manganese-mediated borylation of aryl/heteroaryl diazonium salts emerges as a general and versatile synthetic methodology for the synthesis of the corresponding boronate esters. The reaction proved an ideal testing ground for delineating the Mn species responsible for the photochemical reaction processes, that is, involving either Mn radical or Mn cationic species, which is dependent on the presence of a suitably strong oxidant. Our findings are important for a plethora of processes employing Mn-containing carbonyl species as initiators and/or catalysts, which have considerable potential in synthetic applications.

Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease.

COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments were progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease.

Fragment-derived modulators of an industrial ß-glucosidase.

A fragment screen of a library of 560 commercially available fragments using a kinetic assay identified a small molecule that increased the activity of the fungal glycoside hydrolase TrBgl2. An analogue by catalogue approach and detailed kinetic analysis identified improved compounds that behaved as nonessential activators with up to a 2-fold increase in maximum activation. The compounds did not activate the related bacterial glycoside hydrolase CcBglA demonstrating specificity. Interestingly, an analogue of the initial fragment inhibits both TrBgl2 and CcBglA, apparently through a mixed-model mechanism. Although it was not possible to determine crystal structures of activator binding to 55 kDa TrBgl2, solution NMR experiments demonstrated a specific binding site for the activator. A partial assignment of the NMR spectrum gave the identity of the amino acids at this site, allowing a model for TrBgl2 activation to be built. The activator binds at the entrance of the substrate-binding site, generating a productive conformation for the enzyme-substrate complex.

Design and Synthesis of 56 Shape-Diverse 3D Fragments.

Fragment-based drug discovery is now widely adopted for lead generation in the pharmaceutical industry. However, fragment screening collections are often predominantly populated with flat, 2D molecules. Herein, we describe a workflow for the design and synthesis of 56 3D disubstituted pyrrolidine and piperidine fragments that occupy under-represented areas of fragment space (as demonstrated by a principal moments of inertia (PMI) analysis). A key, and unique, underpinning design feature of this fragment collection is that assessment of fragment shape and conformational diversity (by considering conformations up to 1.5 kcal mol-1 above the energy of the global minimum energy conformer) is carried out prior to synthesis and is also used to select targets for synthesis. The 3D fragments were designed to contain suitable synthetic handles for future fragment elaboration. Finally, by comparing our 3D fragments with six commercial libraries, it is clear that our collection has high three-dimensionality and shape diversity.

Gram-Scale Synthesis of the (-)-Sparteine Surrogate and (-)-Sparteine.

An 8-step, gram-scale synthesis of the (-)-sparteine surrogate (22 % yield, with just 3 chromatographic purifications) and a 10-step, gram-scale synthesis of (-)-sparteine (31 % yield) are reported. Both syntheses proceed with complete diastereocontrol and allow access to either antipode. Since the syntheses do not rely on natural product extraction, our work addresses long-term supply issues relating to these widely used chiral ligands.

Increase of enzyme activity through specific covalent modification with fragments.

Modulation of enzyme activity is a powerful means of probing cellular function and can be exploited for diverse applications. Here, we explore a method of enzyme activation where covalent tethering of a small molecule to an enzyme can increase catalytic activity (kcat/KM) up to 35-fold. Using a bacterial glycoside hydrolase, BtGH84, we demonstrate how small molecule "fragments", identified as activators in free solution, can be covalently tethered to the protein using Michael-addition chemistry. We show how tethering generates a constitutively-activated enzyme-fragment conjugate, which displays both improved catalytic efficiency and increased susceptibility to certain inhibitor classes. Structure guided modifications of the tethered fragment demonstrate how specific interactions between the fragment and the enzyme influence the extent of activation. This work suggests that a similar approach may be used to modulate the activity of enzymes such as to improve catalytic efficiency or increase inhibitor susceptibility.

General Procedures for the Lithiation/Trapping of N-Boc Piperazines.

To provide α-substituted piperazines for early stage medicinal chemistry studies, a simple, general synthetic approach is required. Here, we report the development of two general and simple procedures for the racemic lithiation/trapping of N-Boc piperazines. Optimum lithiation times were determined using in situ IR spectroscopy, and the previous complicated and diverse literature procedures were simplified. Subsequent trapping with electrophiles delivered a wide range of α-functionalized N-Boc piperazines. The scope and limitations of the distal N-group were investigated. The selective α- and ß- arylation of N-Boc piperazines via lithiation/Negishi coupling is reported.

A biosynthesis-inspired approach to over twenty diverse natural product-like scaffolds.

A synthetic approach to diverse scaffolds was developed that was inspired by diterpene biosynthesis. Initial scaffolds, generated using Diels-Alder reactions of furyl-functionalised amines, were transformed into alternative scaffolds using cleavage, ring expansion, annulation and rearrangement reactions. In total, 25 diverse scaffolds were prepared that were shown to have high natural product-likeness.

Synthesis of Enantiopure Piperazines via Asymmetric Lithiation-Trapping of N-Boc Piperazines: Unexpected Role of the Electrophile and Distal N-Substituent.

A new method for the synthesis of enantiopure α-substituted piperazines via direct functionalization of the intact piperazine ring is described. The approach utilizes the asymmetric lithiation-substitution of an α-methylbenzyl-functionalized N-Boc piperazine using s-BuLi/(-)-sparteine or (+)-sparteine surrogate and provides access to a range of piperazines (as single stereoisomers). Optimization of the new methodology required a detailed mechanistic study. Surprisingly, it was found that the main culprits affecting the yield and enantioselectivity were the electrophile (the last reagent to be added to the reaction flask) and the distal N-substituent. The mechanistic studies included optimization of lithiation times using in situ IR spectroscopy, identification of a ring-fragmentation of the lithiated piperazines (that could be minimized with sterically hindered N-alkyl groups), and use of a novel "diamine switch" strategy to improve enantioselectivity with certain electrophiles. The methodology was showcased with the preparation of an intermediate for Indinavir synthesis and the stereoselective synthesis of 2,5-trans- and 2,6-trans-piperazines.

Revisiting the sparteine surrogate: development of a resolution route to the (-)-sparteine surrogate.

The improved performance of the sparteine surrogate compared to sparteine in a range of applications has highlighted the need to develop an approach to the (-)-sparteine surrogate, previously inaccessible in gram-quantities. A multi-gram scale, chromatography-free synthesis of the racemic sparteine surrogate and its resolution via diastereomeric salt formation with (-)-O,O'-di-p-toluoyl-l-tartaric acid is reported. Resolution on a 10.0 mmol scale gave the diastereomeric salts in 33% yield from which (-)-sparteine surrogate of 93 : 7 er was generated. This work solves a key limitation: either enantiomer of the sparteine surrogate can now be readily accessed.

Lipopolysaccharide induces a stromal-epithelial signalling axis in a rat model of chronic periodontitis.

AIM: Lipopolysaccharide is a bacterial virulence factor implicated in chronic periodontitis, which may penetrate the junctional epithelial barrier and basement membrane to insult underlying stroma. We sought to identify lipopolysaccharide-induced global gene expression changes responsible for signalling between stroma and epithelium during disease onset. MATERIALS AND METHODS: Using a rat lipopolysaccharide periodontitis model, junctional epithelium and underlying stromal tissue were separately collected from healthy and diseased animals by laser-capture microdissection and subject to gene expression microarray analysis. Key gene products identified were validated in gingival epithelial and fibroblast cell cultures. RESULTS: Global gene expression patterns distinguishing health versus disease were found in and between both tissue types. In stroma, the most significantly altered gene ontology function group (Z ≥ 4.00) was cytokines, containing most significantly (±2-fold; p < 0.05) upregulated genes amphiregulin, IL1-ß and Fas ligand, all positive, diffusible modulators of the epithelial growth factor receptor pathway. In epithelium, the most significant changes were in downregulated FOS-related antigen-1 gene, somatostatin receptor-2 gene and mucin-4 gene, all negative modulators of the epithelial growth factor receptor pathway. CONCLUSION: These results establish a periodontitis model for studying gene product interactions and suggests that the onset of junctional epithelial disease hyperproliferation involves a concerted stromal-epithelial signalling axis.

An experimental and in situ IR spectroscopic study of the lithiation-substitution of N-Boc-2-phenylpyrrolidine and -piperidine: controlling the formation of quaternary stereocenters.

A general and enantioselective synthesis of 2-substituted 2-phenylpyrrolidines and -piperidines, an important class of pharmaceutically relevant compounds that contain a quaternary stereocenter, has been developed. The approach involves lithiation-substitution of enantioenriched N-Boc-2-phenylpyrrolidine or -piperidine (prepared by asymmetric Negishi arylation or catalytic asymmetric reduction, respectively). The combined use of synthetic experiments and in situ IR spectroscopic monitoring allowed optimum lithiation conditions to be identified: n-BuLi in THF at -50 °C for 5-30 min. Monitoring of the lithiation using in situ IR spectroscopy indicated that the rotation of the tert-butoxycarbonyl (Boc) group is slower in a 2-lithiated pyrrolidine than a 2-lithiated piperidine; low yields for the lithiation-substitution of N-Boc-2-phenylpyrrolidine at -78 °C can be ascribed to this slow rotation. For N-Boc-2-phenylpyrrolidine and -piperidine, the barriers to rotation of the Boc group were determined using density functional theory calculations and variable-temperature (1)H NMR spectroscopy. For the pyrrolidine, the half-life (t(1/2)) for rotation of the Boc group was found to be ∼10 h at -78 °C and ∼3.5 min at -50 °C. In contrast, for the piperidine, t(1/2) was determined to be ∼4 s at -78 °C.

Lipopolysaccharide-induced epithelial monoamine oxidase mediates alveolar bone loss in a rat chronic wound model.

Reactive oxygen species (ROS) production is an antimicrobial response to pathogenic challenge that may, in the case of persistent infection, have deleterious effects on the tissue of origin. A rat periodontal disease model was used to study ROS-induced chronic epithelial inflammation and bone loss. Lipopolysaccharide (LPS) was applied for 8 weeks into the gingival sulcus, and histological analysis confirmed the onset of chronic disease. Junctional epithelium was collected from healthy and diseased animals using laser-capture microdissection, and expression microarray analysis was performed. Of 19,730 genes changed in disease, 42 were up-regulated >/=4-fold. Three of the top 10 LPS-induced genes, monoamine oxidase B (MAO/B) and flavin-containing monooxygenase 1 and 2, are implicated in ROS signaling. LPS-associated induction of the ROS mediator H(2)O(2), as well as MAO/B and tumor necrosis factor (TNF)-alpha levels were validated in the rat histological sections and a porcine junctional epithelial cell culture model. Topical MAO inhibitors significantly counteracted LPS-associated elevation of H(2)O(2) production and TNF-alpha expression in vivo and in vitro, inhibited disease-associated apical migration and proliferation of junctional epithelium and inhibited induced systemic H(2)O(2) levels and alveolar bone loss in vivo. These results suggest that LPS induces chronic wounds via elevated MAO/B-mediated increases in H(2)O(2) and TNF-alpha activity by epithelial cells and is further associated with more distant effects on systemic oxidative stress and alveolar bone loss.

An in vitro analysis of mechanical wounding-induced ligand-independent KGFR activation.

BACKGROUND: KGFR (keratinocyte growth factor receptor), exclusively expressed in epithelial cells, plays an important role in wound healing. However, mechanisms of KGFR activation and signaling in wound healing are not clearly understood. OBJECTIVES: We utilized an in vitro mechanical wounding model to examine ligand-independent KGFR activation, its regulation by reactive oxygen species (ROS) and the functional significance of this activation mechanism. METHODS: Confluent HaCaT cell line cultures were mechanically wounded and KGFR internalization and phosphorylation were examined using immunostaining with confocal microscopy and immunoprecipitation with Western blotting. Wounding-induced generation of reactive oxygen species and ligand-independent activation of KGFR were examined. In addition, phosphorylation of its associated molecules FRS2 and c-Src were examined in the presence and absence of the ROS and pathway specific inhibitors. The importance of this activation process on cell migration was also examined in the presence and absence of these inhibitors. RESULTS: Mechanical wounding induced ligand-independent KGFR activation and internalization. KGFR internalization and phosphorylation was associated with ROS generation along the wound edge and scavenging of ROS with NAC inhibited KGFR phosphorylation. Intracellularly, c-Src was phosphorylated by wounding but its inhibitor, PP1, significantly inhibited KGFR activation and associated FRS2 phosphorylation. Mechanical wounding induced wound edge migration, which was significantly reduced by the selective receptor and pathway inhibitors PP1 (82.7%), KGFR inhibitor SU5402 (70%) and MAPK inhibitor PD98059 (57%). CONCLUSION: Mechanical wounding induces significant ROS generation at the wound edge which, in turn, induced ligand-independent KGFR and FRS2 activation via c-Src kinase signaling. Functionally, downstream MAPK signaling induced wound edge cell migration.

Mechanical induction of an epithelial cell chymase associated with wound edge migration.

Chymase is a chymotrypsin-like serine protease predominantly produced by mast cells. In this study, human cutaneous and gingival keratinocytes, ovary surface epithelia, and a porcine epithelial cell line were assayed by homology-based cloning, and the amplified DNA fragment was identified as a chymase. In vitro, chymase could not be induced by serum or cytokine treatment alone. Chymase was activated 3-fold within 60 min in basal media by scratch wounding cultured monolayers and further potentiated over 10-fold at 18 h by additional serum and cytokine treatment. Chymase activity was cell-associated and found to peak within 24 h of wounding and then steadily decreased as cultures healed, reaching baseline levels before confluence was reestablished. Affinity column purified enzyme effectively degraded fibronectin and was found by Western blot analysis using a human chymase antibody to be of about 30 kDa. Immunostaining revealed chymase activation at the wound edge colocalizing with reactive oxygen species generation. Specifically, chymase activation was attenuated by inhibition of nitric oxide, superoxide, and peroxynitrite. Exogenous peroxynitrite but not hydrogen peroxide also resulted in chymase activation in unwounded monolayers. Disruption of cytoskeletal stress fibers by cytochalasin D attenuated both wound-activated chymase and reactive oxygen species generation. Chymase inhibitor chymostatin reduced the loss of cell-cell contacts and the onset of porcine and human skin epithelial cell migration at the wound edge. This shows that an epithelial chymase is rapidly activated by a ligand-independent mechanism following mechanical stress via cytoskeletal and reactive oxygen species signaling and is associated with the onset of epithelial cell migration.