A thiol-ene mediated approach for peptide bioconjugation using 'green' solvents under continuous flow.

Flow chemistry has emerged as an integral process within the chemical sector permitting energy efficient synthetic scale-up while improving safety and minimising solvent usage. Herein, we report the first applications of the photoactivated, radical-mediated thiol-ene reaction for peptide bioconjugation under continuous flow. Bioconjugation reactions employing deep eutectic solvents, bio-based solvents and fully aqueous systems are reported here for a range of biologically relevant peptide substrates. The use of a water soluble photoinitiator, Irgacure 2959, permitted synthesis of glycosylated peptides in fully aqueous conditions, obviating the need for addition of organic solvents and enhancing the green credentials of these rapid, photoactivated, bioconjugation reactions.

A NIR-Fluorochrome for Live Cell Dual Emission and Lifetime Tracking from the First Plasma Membrane Interaction to Subcellular and Extracellular Locales.

Molecular probes with the ability to differentiate between subcellular variations in acidity levels remain important for the investigation of dynamic cellular processes and functions. In this context, a series of cyclic peptide and PEG bio-conjugated dual near-infrared emissive BF2-azadipyrromethene fluorophores with maxima emissions at 720 nm (at pH > 6) and 790 nm (at pH < 5) have been developed and their aqueous solution photophysical properties determined. Their inter-converting emissions and fluorescence lifetime characteristics were exploited to track their spatial and temporal progression from first contact with the plasma membrane to subcellular locales to their release within extracellular vesicles. A pH-dependent reversible phenolate/phenol interconversion on the fluorophore controlled the dynamic changes in dual emission responses and corresponding lifetime changes. Live-cell confocal microscopy experiments in the metastatic breast cancer cell line MDA-MB-231 confirmed the usability of the dual emissive properties for imaging over prolonged periods. All three derivatives performed as probes capable of real-time continuous imaging of fundamental cellular processes such as plasma membrane interaction, tracking endocytosis, lysosomal/large acidic vesicle accumulation, and efflux within extracellular vesicles without perturbing cellular function. Furthermore, fluorescence lifetime imaging microscopy provided valuable insights regarding fluorophore progression through intracellular microenvironments over time. Overall, the unique photophysical properties of these fluorophores show excellent potential for their use as information-rich probes.

Potential for release of pulmonary toxic ketene from vaping pyrolysis of vitamin E acetate.

A combined analytical, theoretical, and experimental study has shown that the vaping of vitamin E acetate has the potential to produce exceptionally toxic ketene gas, which may be a contributing factor to the upsurge in pulmonary injuries associated with using e-cigarette/vaping products. Additionally, the pyrolysis of vitamin E acetate also produces carcinogen alkenes and benzene for which the negative long-term medical effects are well recognized. As temperatures reached in vaping devices can be equivalent to a laboratory pyrolysis apparatus, the potential for unexpected chemistries to take place on individual components within a vape mixture is high. Educational programs to inform of the danger are now required, as public perception has grown that vaping is not harmful.

Identifying STEDable BF2-Azadipyrromethene Fluorophores.

BF2-azadipyrromethenes are highly versatile fluorophores used for cellular and in vivo imaging in the near-infrared and far-red regions of the spectrum. As of yet, their use in conjunction with super-resolution imaging methodologies has not been explored. In this report, a series of structurally related BF2-azadipyrromethenes has been examined for their suitability for use with stimulated emission depletion (STED) nanoscopy. The potential for STED imaging was initially evaluated using aqueous solutions of fluorophores as an effective predictor of fluorophore suitability. For live cell STED imaging in both 2D and 3D, several far-red emitting BF2-azadipyrromethenes were successfully employed. Image resolution below the diffraction limit of a confocal microscope was demonstrated through measurement of distinct intracellular features including the nuclear membrane, nuclear lamina invaginations, the endoplasmic reticulum, and vacuoles. As the STED ability of BF2-azadipyrromethene fluorophores has now been established, their use with this super-resolution method may be expected to increase in the future.

BF2-Azadipyrromethene Fluorophores for Intraoperative Vital Structure Identification.

A series of mono- and bis-polyethylene glycol (PEG)-substituted BF2-azadipyrromethene fluorophores have been synthesized with emissions in the near-infrared region (700-800 nm) for the purpose of fluorescence guided intraoperative imaging; chiefly ureter imaging. The Bis-PEGylation of fluorophores resulted in higher aqueous fluorescence quantum yields, with PEG chain lengths of 2.9 to 4.6 kDa being optimal. Fluorescence ureter identification was possible in a rodent model with the preference for renal excretion notable through comparative fluorescence intensities from the ureters, kidneys and liver. Ureteral identification was also successfully performed in a larger animal porcine model under abdominal surgical conditions. Three tested doses of 0.5, 0.25 and 0.1 mg/kg all successfully identified fluorescent ureters within 20 min of administration which was sustained up to 120 min. 3-D emission heat map imaging allowed the spatial and temporal changes in intensity due to the distinctive peristaltic waves of urine being transferred from the kidneys to the bladder to be identified. As the emission of these fluorophores could be spectrally distinguished from the clinically-used perfusion dye indocyanine green, it is envisaged that their combined use could be a step towards intraoperative colour coding of different tissues.

Continuous Flow Bioconjugations of NIR-AZA Fluorophores via Strained Alkyne Cycloadditions with Intra-Chip Fluorogenic Monitoring.

The importance of bioconjugation reactions continues to grow for cell specific targeting and dual therapeutic plus diagnostic medical applications. This necessitates the development of new bioconjugation chemistries, in-flow synthetic and analytical methods. With this goal, continuous flow bioconjugations were readily achieved with short residence times for strained alkyne substituted carbohydrate and therapeutic peptide biomolecules in reaction with azide and tetrazine substituted fluorophores. The strained alkyne substrates included substituted 2-amino-2-deoxy-α-D-glucopyranose, and the linear and cyclic peptide sequences QIRQQPRDPPTETLELEVSPDPAS-OH and c(RGDfK) respectively. The catalyst and reagent-free inverse electron demand tetrazine cycloadditions proved more favourable than the azide 1,3-dipolar cycloadditions. Reaction completion was achieved with residence times of 5 min at 40 °C for tetrazine versus 10 min at 80 °C for azide cycloadditions. The use of a fluorogenic tetrazine fluorophore, in a glass channelled reactor chip, allowed for intra-chip reaction monitoring by recording fluorescence intensities at various positions throughout the chip. As the Diels-Alder reactions proceeded through the chip, the fluorescence intensity increased accordingly in real-time. The application of continuous flow fluorogenic bioconjugations could offer an efficient translational access to theranostic agents.

Dual Color Imaging from a Single BF2-Azadipyrromethene Fluorophore Demonstrated in vivo for Lymph Node Identification.

Dual emissions at ~700 and 800 nm have been achieved from a single NIR-AZA fluorophore 1 by establishing parameters in which it can exist in either its isolated molecular or aggregated states. Dual near infrared (NIR) fluorescence color lymph node (LN) mapping with 1 was achieved in a large-animal porcine model, with injection site, channels and nodes all detectable at both 700 and 800 nm using a preclinical open camera system. The fluorophore was also compatible with imaging using two clinical instruments for fluorescence guided surgery. Methods: An NIR-AZA fluorophore with hydrophilic and phobic features was synthesised in a straightforward manner and its aggregation properties characterised spectroscopically and by TEM imaging. Toxicity was assessed in a rodent model and dual color fluorescence imaging evaluated by lymph node mapping in a large animal porcine models and in ex-vivo human tissue specimen. Results: Dual color fluorescence imaging has been achieved in the highly complex biomedical scenario of lymph node mapping. Emissions at 700 and 800 nm can be achieved from a single fluorophore by establishing molecular and aggregate forms. Fluorophore was compatible with clinical systems for fluorescence guided surgery and no toxicity was observed in high dosage testing. Conclusion: A new, biomedical compatible form of NIR-dual emission wavelength imaging has been established using a readily accessible fluorophore with significant scope for clinical translation.

Aminopotassiation by Mixed Potassium/Lithium Amides: A Synthetic Path to Difficult to Access Phenethylamine Derivates.

Insights gained from a comparison of aminometalation reactions with lithium amides, potassium amides and mixed lithium/potassium amides are presented. A combination of structural characterization, DFT calculations and electrophile reactions of aminometalated intermediates has shown the advantages of using a mixed metal strategy. While potassium amides fail to add, the lithium amides are uncontrollable and eliminated, yet the mixed K/Li amides deliver the best of both systems. Aminopotassiation proceeds to form the alkylpotassium species which has enhanced stability over its lithium counterpart allowing for its isolation and thereby its further characterization.

Multimodal Microscopy Distinguishes Extracellular Aggregation and Cellular Uptake of Single-Walled Carbon Nanohorns.

The low toxicity, high surface area, and ease of functionalisation of carbon nanohorns (CNH) makes them attractive systems for cellular imaging, diagnostics and therapeutics. However, challenges remain for the biomedical translation of these and other nanomaterials. A significant task is tuning the surface chemistry to achieve optimal cellular interactions. Herein, we combine real-time fluorescent imaging of nanoparticle cellular uptake and real-time differential interference contrast (DIC) imaging of extracellular media to monitor a) nanoparticle/nanoparticle and b) nanoparticle/cell interactions for CNHs covalently modified with an OFF/ON near-IR dye, the fluorescence of which is switched OFF in extracellular environments and triggered upon cellular internalisation. CHN samples modified with different loadings of the hydrophobic dye are taken as a simple model of drug-loaded nanoparticle systems. The punctate fluorescence suggests the CNHs are delivered to lysosomes and other vesicles of the endocytic pathway. DIC imaging highlights the competition that exists for many particle types, between extracellular aggregation and cellular internalization, the efficiency of which would be dependent upon the amount of fluorophore loading. The results of this study illustrate how complementary real-time imaging methods together with physicochemical characterisation can be used to address the challenges involved in optimising nanoparticle/cell interactions for biomedical applications.

Azadipyrromethenes: from traditional dye chemistry to leading edge applications.

Azadipyrromethenes were first described over 70 years ago as blue pigments, but now are rapidly emerging as a compound class with highly desirable near infrared photophysical properties. Since the turn of the century several routes to azadipyrromethenes have been developed and numerous post-synthesis derivatizations have allowed for their exploitation in both biological and material sciences. The relative ease of access to specifically designed derivatives is now allowing their use in multiple technological formats from real-time fluorescence imaging, to solar energy materials, to optoelectronic devices and many more. In this review we have highlighted the synthetic component of this story as it is the ability to generate the designer azadipyrromethene that opens the door to exciting applications.

Highly Selective Addition of a Broad Spectrum of Trimethylsilane Pro-nucleophiles to N-tert-Butanesulfinyl Imines.

Addition of organotrimethylsilane reagents to chiral N-tert-butanesulfinyl imines can be achieved in good yields and with excellent diastereoselectivities by employing TMSO(-)/Bu4N(+) as a Lewis base activator in THF. A variety of aliphatic, aromatic, heteroaromatic and organometallic chiral imines were utilised as electrophiles for the synthesis of enantioenriched N-tert-butanesulfinyl amides. Remarkably, the same sets of reaction conditions could be used with a highly diverse range of bench-stable organotrimethylsilane reagents, which highlights the generality and robustness of this methodology.

Stereoselective Peterson olefinations from bench-stable reagents and N-phenyl imines.

The synthesis of bench-stable α,α-bis(trimethylsilyl)toluenes and tris(trimethylsilyl)methane is described and their use in stereoselective Peterson olefinations has been achieved with a wide substrate scope. Product stereoselectivity was poor with carbonyl electrophiles (E/Z ∼1:1 to 4:1) though this was significantly improved by employing the corresponding substituted N-benzylideneaniline (up to 99:1) as an alternative electrophile. The olefination byproduct was identified as N,N-bis(trimethylsilyl)aniline and could be easily separated from product by aqueous acid extraction. Evidence for an autocatalytic cycle has been obtained.

General Ambient Temperature Benzylic Metalations Using Mixed-Metal Li/K-TMP Amide.

Highly regioselective benzylic metalations in hydrocarbon solvent have been achieved at rt and 0 °C using a mixed-metal Li/K-TMP amide comprised of KOtBu, BuLi, and 2,2,6,6,-tetramethylpiperidine (TMP(H)). Mixing of KOtBu, BuLi, and TMP(H) in heptane gave a solution of the base mixture which when used in deuterium labeling experiments confirmed the requirement of the three reagent components for both reactivity and selectivity. The reaction protocol is operationally straightforward and found to be applicable to a broad range of substrates. Upon generation of the metalated products, they are reacted in heptane at ambient temperature in a variety of synthetically useful ways. Illustrated examples include generation of the benzyltrimethylsilanes and α,α-bis(trimethylsilyl)toluenes reagents, which are bench-stable surrogates of benzyl anions and α-silyl carbanions utilized for nucleophilic addition and Peterson olefination reactions. Direct C-C couplings mediated by 1,2-dibromoethane provided entries into bibenzyls and [2.2]metacyclophanes. Comparison of reaction outcomes with the same reactions carried out in THF at -78 °C showed no negative effects for conducting the reactions under these milder more user-friendly conditions.

Bu4N+ alkoxide-initiated/autocatalytic addition reactions with organotrimethylsilanes.

The use of Me3SiO(-)/Bu4N(+) as a general activator of organotrimethylsilanes for addition reactions has been established. The broad scope of the method offers trimethylsilanes (including acetate, allyl, propargyl, benzyl, dithiane, heteroaryl, and aryl derivatives) as bench-stable organometallics that can be readily utilized as carbanion equivalents for synthesis. Reactions are achieved at rt without the requirement of specialized precautions that are commonplace for other organometallics.

Insights into the metalation of benzene and toluene by Schlosser's base: a superbasic cluster comprising PhK, PhLi, and tBuOLi.

The metalation of benzene by Schlosser's base (nBuLi/tBuOK) occurs smoothly in THF at low temperatures to afford a discrete mixed-metal Li2 K4 cluster that contains phenyl anions and tert-butoxide. The aggregate itself exhibits superbasic behavior by metalating toluene. The delocalized benzyl anion obtained this way π bonds to potassium counterions, thereby creating a 2D coordination polymer.

Forecasting vaping health risks through neural network model prediction of flavour pyrolysis reactions.

Vaping involves the heating of chemical solutions (e-liquids) to high temperatures prior to lung inhalation. A risk exists that these chemicals undergo thermal decomposition to new chemical entities, the composition and health implications of which are largely unknown. To address this concern, a graph-convolutional neural network (NN) model was used to predict pyrolysis reactivity of 180 e-liquid chemical flavours. The output of this supervised machine learning approach was a dataset of probability ranked pyrolysis transformations and their associated 7307 products. To refine this dataset, the molecular weight of each NN predicted product was automatically correlated with experimental mass spectrometry (MS) fragmentation data for each flavour chemical. This blending of deep learning methods with experimental MS data identified 1169 molecular weight matches that prioritized these compounds for further analysis. The average number of discrete matches per flavour between NN predictions and MS fragmentation was 6.4 with 92.8% of flavours having at least one match. Globally harmonized system classifications for NN/MS matches were extracted from PubChem, revealing that 127 acute toxic, 153 health hazard and 225 irritant classifications were predicted. This approach may reveal the longer-term health risks of vaping in advance of clinical diseases emerging in the general population.

Synthesis of trisubstituted alkenes via direct oxidative arene-alkene coupling.

The use of an inorganic oxidant with an acetic acid/acetonitrile solvent combination has been identified as optimal for direct arene/1,2-disubstituted alkene oxidative couplings, providing an efficient route to trisubstituted alkenes. The acetonitrile cosolvent dramatically accelerates the rate of reaction, and an insoluble inorganic oxidant limits unwanted oxidation of substrates. The scope of this procedure is illustrated with arenes and alkenes containing electron-donating and -withdrawing substituents resulting in a general synthetic strategy to trisubstituted alkenes. In situ ESI-MS analysis of the reaction components has identified the key Pd intermediates in the Fujiwara-Moritani catalytic cycle.

Substituent directed cellular imaging in the 800-850 nm range with BF2-azadipyrromethene fluorophores.

Three bis(anilino)-substituted NIR-AZA fluorophores have been designed, synthesized and tested to bridge the availability gap of molecular fluorophores for live-cell microscopy imaging in the 800-850 nm spectral range. The concise synthetic route allows for the later stage introduction of three tailored peripheral substituents which guides the sub-cellular localization and imaging. Live-cell fluorescence imaging of lipid droplets, plasma membrane and cytosolic vacuoles was successfully achieved. Photophysical and internal charge transfer (ICT) properties of each fluorophore were examined through solvent studies and analyte responses.