Susceptibility of the Different Oxygen-Sensing Probes to Interferences in Respirometric Bacterial Assays with Complex Media.

Respirometric microbial assays are gaining popularity, but their uptake is limited by the availability of optimal O2 sensing materials and the challenge of validating assays with complex real samples. We conducted a comparative evaluation of four different O2-sensing probes based on Pt-porphyrin phosphors in respirometric bacterial assays performed on standard time-resolved fluorescence reader. The macromolecular MitoXpress, nanoparticle NanO2 and small molecule PtGlc4 and PtPEG4 probes were assessed with E. coli cells in five growth media: nutrient broth (NB), McConkey (MC), Rapid Coliform ChromoSelect (RCC), M-Lauryl lauryl sulfate (MLS), and Minerals-Modified Glutamate (MMG) media. Respiration profiles of the cells were recorded and analyzed, along with densitometry profiles and quenching studies of individual media components. This revealed several limiting factors and interferences impacting assay performance, which include probe quenched lifetime, instrument temporal resolution, inner filter effects (mainly by indicator dyes), probe binding to lipophilic components, and dynamic and static quenching by media components. The study allowed for the ranking of the probes based on their ruggedness, resilience to interferences and overall performance in respirometric bacterial assays. The 'shielded' probe NanO2 outperformed the established MitoXpress probe and the small molecule probes PtGlc4 and PtPEG4.

Flow photolysis of aryldiazoacetates leading to dihydrobenzofurans via intramolecular C-H insertion.

Flow photolysis of aryldiazoacetates 3-5 leads to C-H insertion to form dihydrobenzofurans 6-8 in a metal-free process, using either a medium pressure mercury lamp (250-390 nm) or LEDs (365 nm or 450 nm) with comparable synthetic outcomes. Significantly, addition of 4,4'-dimethoxybenzophenone 9 results in an increased yield and also alters the stereochemical outcome leading to preferential isolation of the trans dihydrobenzofurans 6a-8a (up to 50% yield), while the cis and trans diastereomers of 6-8 are recovered in essentially equimolar amounts in the absence of a photosensitiser (up to 26% yield).

Heterosubstituted Derivatives of PtPFPP for O2 Sensing and Cell Analysis: Structure-Activity Relationships.

Biological applications of phosphorescent probes for sensing molecular oxygen (O2) and bioimaging have gained popularity, but their choice is rather limited. We describe a family of new heterosubstituted phosphorescent bioprobes based on the Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP) dye. The probes are produced by simple click modification of its para-fluorine atoms with thiols, such as 1/2-thio-glucose, thio-poly(ethylene glycol) (PEG), or cysteamine. The probes were designed to have one cell-targeting moiety and three polar moieties forming a hydrophilic shell. Their chemical synthesis and purification were optimized to produce high reaction yields and easy scale-up. The ability to perform as cell-permeable or -impermeable probes was tuned by the polarity and molecular charge of the bioconjugate. The new PtPFPP derivatives were characterized for their spectral properties and cell-penetrating ability in the experiments with mammalian cell cultures, using a time-resolved fluorescence reader and PLIM imaging detection. Structure-activity relationships were established. Thus, the tri- and tetra-PEGylated structures showed low cell internalization allowing their use as extracellular probes, while cysteamine derivatives performed as efficient intracellular probes. No significant cytotoxicity was observed for all of the probes under the experimental conditions used.

Synthesis and Evaluation of Prodrugs of α-Carboxy Nucleoside Phosphonates.

A range of lipophilic prodrugs of α-carboxy nucleoside phosphonates, potent inhibitors of HIV-1 reverse transcriptase without requiring prior phosphorylation, were synthesized to evaluate their in vivo potency against HIV in cell culture. A series of prodrug derivatives bearing a free carboxylic acid where the phosphonate was masked with bispivaloyloxymethyl, diisopropyloxycarbonyloxymethyl, bisamidate, aryloxyphosphoramidate, hexadecyloxypropyl, CycloSal, and acycloxybenzyl moieties were synthesized, adapting existing methodologies for phosphonate protection to accommodate the adjacent carboxylic acid moiety. The prodrugs were assayed for anti-HIV activity in CEM cell cultures─the bispivaloyloxymethyl free acid monophosphonate prodrug exhibited some activity (inhibitory concentration-50 (IC50) 59 ± 17 µM), while the other prodrugs were inactive at 100 µM. A racemic bispivaloyloxymethyl methyl ester monophosphonate prodrug was also prepared to assess the suitability of the methyl ester as a carboxylic acid prodrug. This compound exhibited no activity against HIV in cellular assays.

Exploiting Continuous Processing for Challenging Diazo Transfer and Telescoped Copper-Catalyzed Asymmetric Transformations.

Generation and use of triflyl azide in flow enables efficient synthesis of a range of α-diazocarbonyl compounds, including α-diazoketones, α-diazoamides, and an α-diazosulfonyl ester, via both Regitz-type diazo transfer and deacylative/debenzoylative diazo-transfer processes with excellent yields and offers versatility in the solvent employed, in addition to addressing the hazards associated with handling of this highly reactive sulfonyl azide. Telescoping the generation of triflyl azide and diazo-transfer process with highly enantioselective copper-mediated intramolecular aromatic addition and C-H insertion processes demonstrates that the reaction stream containing the α-diazocarbonyl compound can be obtained in sufficient purity to pass directly over the immobilized copper bis(oxazoline) catalyst without detrimentally impacting the catalyst enantioselectivity.

Exploring the synthetic potential of a marine transaminase including discrimination at a remote stereocentre.

The marine transaminase, P-ω-TA, can be employed for the transamination from 1-aminotetralins and 1-aminoindanes with differentiation of stereochemistry at both the site of reaction and at a remote stereocentre resulting in formation of ketone products with up to 93% ee. While 4-substituents are tolerated on the tetralin core, the presence of 3- or 8-substituents is not tolerated by the transaminase. In general P-ω-TA shows capacity for remote diastereoselectivity, although both the stereoselectivity and efficiency are dependent on the specific substrate structure. Optimum efficiency and selectivity are seen with 4-haloaryl-1-aminotetralins and 3-haloaryl-1-aminoindanes, which may be associated with the marine origin of this enzyme.

Localized Partitioning of Enantiomers in Solid Samples of Sulfoxides: Importance of Sampling Method in Determination of Enantiopurity.

Localized partitioning of amorphous enantioenriched aryl benzyl sulfoxides in the solid state can lead to substantial variation in enantiopurities, even for sulfoxides which do not show detectable levels of self-disproportionation of enantiomers (SDE) during chromatography on an achiral stationary phase. The importance of preparation of representative samples of enantioenriched sulfoxides for chiral HPLC to enable reproducible results is clear.

Synthetic and mechanistic aspects of sulfonyl migrations.

Over the past 20 years reports of sulfonyl migrations have appeared, frequently described as 'unusual' and 'unexpected'. This comprehensive review compiles, for the first time, sulfonyl migrations reported over the last 20 years including formal 1,2-, 1,3-, 1,4-, 1,5-, 1,6- and 1,7-sulfonyl shifts, occurring through either radical or polar processes, either inter- or intramolecularly. Discussion of the sulfonyl migrations is structured according to reaction type, i.e. nitrogen-carbon, nitrogen-oxygen, nitrogen-nitrogen, oxygen-carbon (including anionic and non-anionic thia-Fries rearrangements), oxygen-oxygen and carbon-carbon migrations. Discussion of the underlying mechanisms for the migrations is included, with particular attention afforded to the principal techniques utilised for their elucidation, namely isotopic-labelling, crossover experiments, density functional theory calculations and electron paramagnetic resonance spectroscopy amongst others.

Desymmetrization by Asymmetric Copper-Catalyzed Intramolecular C-H Insertion Reactions of α-Diazo-ß-oxosulfones.

Effective desymmetrization in copper-catalyzed intramolecular C-H insertion reactions of α-diazo-ß-oxosulfones in the formation of fused thiopyran dioxides is described for the first time. The use of a copper-bis(oxazoline)-NaBARF catalyst complex system leads to formation of the major thiopyran dioxide stereoisomer with up to 98:2 dr and up to 98% ee. The effect of varying the bis(oxazoline) ligand, copper salt, and site of C-H insertion on both diastereo- and enantioselectivities of these intramolecular C-H insertion reactions has been investigated. Similarly, desymmetrization in the formation of a fused cyclopentanone proceeds with up to 64% ee. These results represent the highest enantioselectivity reported to date in a copper-mediated desymmetrization through C-H insertion.

Correction: Enantioselective copper catalysed intramolecular C-H insertion reactions of α-diazo-ß-keto sulfones, α-diazo-ß-keto phosphine oxides and 2-diazo-1,3-diketones; the influence of the carbene substituent.

Correction for 'Enantioselective copper catalysed intramolecular C-H insertion reactions of α-diazo-ß-keto sulfones, α-diazo-ß-keto phosphine oxides and 2-diazo-1,3-diketones; the influence of the carbene substituent' by Amy E. Shiely et al., Org. Biomol. Chem., 2017, 15, 2609-2628.

Synthesis of 1,2,5-oxathiazole-S-oxides by 1,3-dipolar cycloadditions of nitrile oxides to α-oxo sulfines.

Synthetic methodology for the generation of novel 1,2,5-oxathiazole-S-oxides from cycloaddition of nitrile oxide dipoles with α-oxo sulfines generated in situ via the α-sulfinyl carbenes derived from α-diazosulfoxides is described. Experimental evidence and mechanistic rationale for the unanticipated interconversion of the diastereomeric 1,2,5-oxathiazole-S-oxide cycloadducts are discussed. Notably, using rhodium acetate as a catalyst at 0 °C under traditional batch conditions led to the selective formation and isolation of the kinetic isomers, while, in contrast, using continuous flow thermolysis, optimal conditions for the synthesis and isolation of the thermodynamic isomers were established.

Industry-Academia Partnership: The Synthesis and Solid-State Pharmaceutical Centre (SSPC) as a Collaborative Approach from Molecule to Medicine.

A marriage made in Ireland! The Synthesis and Solid-State Pharmaceutical Centre (SSPC) transcends company and academic boundaries and is one of the largest research collaborations of its type globally. Trust, a culture of inclusivity and commitment to mutual benefit are just some of the factors that have led to the success of the SSPC. Fostering these ideals within its industry-academia, and inter-company collaborations remains crucial to further development.

Copper-Mediated, Heterogeneous, Enantioselective Intramolecular Buchner Reactions of α-Diazoketones Using Continuous Flow Processing.

Enantioselective intramolecular Buchner reactions of α-diazoketones can be effected using heterogeneous copper-bis(oxazoline) catalysts in batch or using continuous flow processing in up to 83% ee. The catalyst can be reused up to 7 times without loss of activity. For α-diazoketones 3 and 4, the enantioselection achieved in flow with the immobilized catalyst was comparable with the standard homogeneous catalyzed process.

Synthesis of Guanine α-Carboxy Nucleoside Phosphonate (G-α-CNP), a Direct Inhibitor of Multiple Viral DNA Polymerases.

The synthesis of guanine α-carboxy nucleoside phosphonate (G-α-CNP) is described. Two routes provide access to racemic G-α-CNP 9, one via base construction and the other utilizing Tsuji-Trost allylic substitution. The latter methodology was also applied to the enantiopure synthesis of both antipodes of G-α-CNP, each of which showing interesting antiviral DNA polymerase activity. Additionally, we report an improved multigram scale preparation of the cyclopentene building block 10, starting material for the preferred Tsuji-Trost route to 9.

Alpha-carboxy nucleoside phosphonates as universal nucleoside triphosphate mimics.

Polymerases have a structurally highly conserved negatively charged amino acid motif that is strictly required for Mg(2+) cation-dependent catalytic incorporation of (d)NTP nucleotides into nucleic acids. Based on these characteristics, a nucleoside monophosphonate scaffold, α-carboxy nucleoside phosphonate (α-CNP), was designed that is recognized by a variety of polymerases. Kinetic, biochemical, and crystallographic studies with HIV-1 reverse transcriptase revealed that α-CNPs mimic the dNTP binding through a carboxylate oxygen, two phosphonate oxygens, and base-pairing with the template. In particular, the carboxyl oxygen of the α-CNP acts as the potential equivalent of the α-phosphate oxygen of dNTPs and two oxygens of the phosphonate group of the α-CNP chelate Mg(2+), mimicking the chelation by the ß- and γ-phosphate oxygens of dNTPs. α-CNPs (i) do not require metabolic activation (phosphorylation), (ii) bind directly to the substrate-binding site, (iii) chelate one of the two active site Mg(2+) ions, and (iv) reversibly inhibit the polymerase catalytic activity without being incorporated into nucleic acids. In addition, α-CNPs were also found to selectively interact with regulatory (i.e., allosteric) Mg(2+)-dNTP-binding sites of nucleos(t)ide-metabolizing enzymes susceptible to metabolic regulation. α-CNPs represent an entirely novel and broad technological platform for the development of specific substrate active- or regulatory-site inhibitors with therapeutic potential.

Synthesis of Cyclic α-Diazo-ß-keto Sulfoxides in Batch and Continuous Flow.

Diazo transfer to ß-keto sulfoxides to form stable isolable α-diazo-ß-keto sulfoxides has been achieved for the first time. Both monocyclic and benzofused ketone derived ß-keto sulfoxides were successfully explored as substrates for diazo transfer. Use of continuous flow leads to isolation of the desired compounds in enhanced yields relative to standard batch conditions, with short reaction times, increased safety profile, and potential to scale up.

Enantioselective copper catalysed intramolecular C-H insertion reactions of α-diazo-ß-keto sulfones, α-diazo-ß-keto phosphine oxides and 2-diazo-1,3-diketones; the influence of the carbene substituent.

Enantioselectivities in C-H insertion reactions, employing the copper-bis(oxazoline)-NaBARF catalyst system, leading to cyclopentanones are highest with sulfonyl substituents on the carbene carbon, and furthermore, the impact is enhanced by increased steric demand on the sulfonyl substituent (up to 91%ee). Enantioselective intramolecular C-H insertion reactions of α-diazo-ß-keto phosphine oxides and 2-diazo-1,3-diketones are reported for the first time.

Taming tosyl azide: the development of a scalable continuous diazo transfer process.

Heat and shock sensitive tosyl azide was generated and used on demand in a telescoped diazo transfer process. Small quantities of tosyl azide were accessed in a 'one pot' batch procedure using shelf stable, readily available reagents. For large scale diazo transfer reactions tosyl azide was generated and used in a telescoped flow process, to mitigate the risks associated with handling potentially explosive reagents on scale. The in situ formed tosyl azide was used to rapidly perform diazo transfer to a range of acceptors, including ß-ketoesters, ß-ketoamides, malonate esters and ß-ketosulfones. An effective in-line quench of sulfonyl azides was also developed, whereby a sacrificial acceptor molecule ensured complete consumption of any residual hazardous diazo transfer reagent. The telescoped diazo transfer process with in-line quenching was used to safely prepare over 21 g of an α-diazocarbonyl in >98% purity without any column chromatography.

Exploring the role of the α-carboxyphosphonate moiety in the HIV-RT activity of α-carboxy nucleoside phosphonates.

As α-carboxy nucleoside phosphonates (α-CNPs) have demonstrated a novel mode of action of HIV-1 reverse transcriptase inhibition, structurally related derivatives were synthesized, namely the malonate 2, the unsaturated and saturated bisphosphonates 3 and 4, respectively and the amide 5. These compounds were evaluated for inhibition of HIV-1 reverse transcriptase in cell-free assays. The importance of the α-carboxy phosphonoacetic acid moiety for achieving reverse transcriptase inhibition, without the need for prior phosphorylation, was confirmed. The malonate derivative 2 was less active by two orders of magnitude than the original α-CNPs, while displaying the same pattern of kinetic behavior; interestingly the activity resides in the "L"-enantiomer of 2, as seen with the earlier series of α-CNPs. A crystal structure with an RT/DNA complex at 2.95 Å resolution revealed the binding of the "L"-enantiomer of 2, at the polymerase active site with a weaker metal ion chelation environment compared to 1a (T-α-CNP) which may explain the lower inhibitory activity of 2.

Development of a continuous process for α-thio-ß-chloroacrylamide synthesis with enhanced control of a cascade transformation.

A continuous process strategy has been developed for the preparation of α-thio-ß-chloroacrylamides, a class of highly versatile synthetic intermediates. Flow platforms to generate the α-chloroamide and α-thioamide precursors were successfully adopted, progressing from the previously employed batch chemistry, and in both instances afford a readily scalable methodology. The implementation of the key α-thio-ß-chloroacrylamide casade as a continuous flow reaction on a multi-gram scale is described, while the tuneable nature of the cascade, facilitated by continuous processing, is highlighted by selective generation of established intermediates and byproducts.