Microscale Reactions Research Articles

Ultra-intense femtosecond laser interactions with aligned nanostructures

The interaction of ultrafast laser pulses of relativistic intensity with high aspect ratio nanostructures can efficiently and volumetrically heat matter to an ultra-high-energy-density regime encountered in the center of stars and within the core of fusion capsules compressed by the world’s largest lasers. It also generates gigantic quasi-static electromagnetic fields that accelerate particles to very high energy. Here, we present an overview of the physics and applications of these dense relativistic plasmas that can be created with pulses of relatively modest energy from lasers that can operate at a high repetition rate. Recent nanowire array experiments produced near-solid density plasmas with an extreme degree of ionization (e.g., Au+72), converted ultrafast pulses of laser light into intense x-ray flashes with record efficiency, and accelerated ions to MeV energies, efficiently driving micro-scale fusion reactions that generate flashes of quasi-monoenergetic neutrons. These plasmas also serve as a platform for advancing the understanding of atomic processes in extreme environments and open a new pathway to laser-driven fusion energy. The irradiation of nanostructures at intensities of >1×1022Wcm−2 is predicted to lead to an extreme ultra-high energy density plasma regime characterized by terabar pressures that is virtually unexplored.

Development of Prodrug-Payloads for Targeted Therapeutic Applications of Platinum-Acridine Anticancer Agents.

A synthetic platform has been developed that provides access to platinum(IV) prodrugs of highly cytotoxic platinum-acridine anticancer agents and allows them to be incorporated into conjugation-ready prodrug-payloads (PPLs). The PPLs can be conveniently assembled in highly efficient microscale reactions utilizing strain-promoted azide-alkyne cycloaddition chemistry. Model reactions were performed to study the stability of the PPLs in buffers and media and to assess their compatibility with cysteine-maleimide Michael addition chemistry. Amide coupling was a successful strategy to generate a conjugate containing integrin-targeted cyclo[RGDfK] peptide. Reactions with ascorbate were performed to mimic the reductive activation of the PPLs and the latter conjugate, and a cyanine (Cy5) fluorophore-labeled PPL was used to probe the reduction of platinum(IV) in cancer cells by confocal microscopy. The PPL concept introduced here should be evaluated for treating solid tumors with PAs using cancer-targeting vehicles, such as antibody-drug conjugates.

Enhanced scale-up performance on residence time distribution by integrated microcapillaries with high size uniformity

Continuous tubular flow reactors are widely used by virtue of their simple structures, controllable reaction conditions, enhanced heat and mass transfer, etc. Microreactors present specific microscale characteristics including laminar flow, reduced diffusion path, enhanced mixing degree and high surface area to volume ratio. Microcapillary reactors combined the advantages of continuous processing and microscale reactions. However, the scale-up of microreactions is still a very challenging task for the loss of enhanced transport characteristics in microscale. Residence time distribution (RTD) is a vital research method to investigate the flow behaviors of microreactors. Microcapillary films (MCFs) containing several parallel microcapillaries provide a strategy to multiplex the microreactions. Nevertheless, the low size uniformity of microcapillaries in MCF strips hinders their application in microreactions. We carried out Computational Fluid Dynamic (CFD) simulations with experimental RTD validation to investigate the effects of dimension, convection and diffusion on RTD and the relationship between the RTD diversity and the size uniformity of microcapillaries under fixed velocities and volumetric flow rates. MCFs with high size uniformity were manufactured by liquid-assisted extrusion, and the coefficient of variance of the mean residence times of the microcapillaries in the MCF strip was only 2.8%. Neutralization reaction also showed the consistence of reaction performances among microcapillaries. With the enhanced scale-up performance on RTD and microreactions, MCFs are expected to have broad applications in large-scale microreactions in chemical engineering industry.

Streptomyces cell-free systems for natural product discovery and engineering.

Streptomyces bacteria are a major microbial source of natural products, which are encoded within so-called biosynthetic gene clusters (BGCs). This highlight discusses the emergence of native Streptomyces cell-free systems as a new tool to accelerate the study of the fundamental chemistry and biology of natural product biosynthesis from these bacteria. Cell-free systems provide a prototyping platform to study plug-and-play reactions in microscale reactions. So far, Streptomyces cell-free systems have been used to rapidly characterise gene expression regulation, access secondary metabolite biosynthetic enzymes, and catalyse cell-free transcription, translation, and biosynthesis of example natural products. With further progress, we anticipate the development of more complex systems to complement existing experimental tools for the discovery and engineering of natural product biosynthesis from Streptomyces and related high G + C (%) bacteria.

Numerical characterization of inter-core coalescence by AC dielectrophoresis in double-emulsion droplets.

The utilization of an alternating current electric field provides a good means to achieve controlled coalescence between paired inner cores encapsulated in water-in-oil-in-water double-emulsion (DE) droplets. Although previous studies have experimentally determined the conditions under which inter-core electrokinetic fusion occurs, the transient interfacial dielectrophoretic (DEP) dynamics key to understand the underlying fluid mechanics is still unclear from a physical point of view. By coupling DEP motion of two-phase flow to phase-field formulation, bulk-coupled numerical simulations are conducted to characterize the spatial-temporal evolution of the surface charge wave and the resulting nonlinear electrical force induced at both the core/shell and medium/shell oil/water interfaces. The effect of interfacial charge relaxation and droplet geometry on inter-core attractive dipolar interaction is investigated within a wide parametric space, and four distinct device operation modes, including normal inter-core fusion, shell elongation, partial core leakage, and complete core release, are well distinguished from one another by flow regime argumentation. Our results herein reveal for the first time the hitherto unknown transient electrohydrodynamic fluid motion of DE droplet driven by Maxwell-Wagner structural polarization. The dynamic simulation method proposed in present study points out an effective outlet to predict the nonlinear electrokinetic behavior of multicore DE droplets for realizing a more controlled triggering of microscale reactions for a wide range of applications in drug discovery, skin care, and food industry.

Hot spot ignition and growth from tandem micro-scale simulations and experiments on plastic-bonded explosives

Head-to-head comparisons of multiple experimental observations and numerical simulations on a deconstructed plastic-bonded explosive consisting of an octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine crystal embedded in a polymeric binder with a 4 ns duration 20 GPa input shock are presented. Hot spots observed in high-resolution direct numerical simulations are compared with micro-scale shock-induced reactions visualized using nanosecond microscope imaging and optical pyrometry. Despite the challenges and limitations of both the experimental and simulation techniques, an agreement is obtained on many of the observed features of hot spot evolution, e.g., (1) the magnitude and time variation of temperatures in the hot spots, (2) the time to fully consume the crystals (∼100 ns) of size (100–300 μm) employed in this study, and (3) the locations of hot spot initiation and growth. Three different mechanisms of hot spot formation are indicated by simulations: (1) high-temperature hot spots formed by pore collapse, (2) lower temperature hot spots initiated at the polymer–crystal interface near corners and asperities, and (3) high-temperature reaction waves leading to fast consumption of the energetic crystal. This first attempt at a head-to-head comparison between experiments and simulations not only provides new insight but also highlights efforts needed to bring models and experiments into closer alignment, in particular, highlighting the importance of distinctly three-dimensional and multiple mechanisms of the hot spot ignition and growth.

Indicators as Mediators for Environmental Decision Making: The Case Study of Alessandria

The design of urban public open spaces plays a key role in the development of micro-scale reactions to global phenomena (pandemic, climate change, etc.) that are currently reshaping the human habitat. Their transformability and healthy influence on the urban environment make them strategic nodes for acupunctural regeneration with systemic effects. Several methods, models, and indicators have been developed to face the complexity of these spaces, made up of tangible and intangible layers; however, there is a gap between theoretical investigation and the need for public administrations to devise feasible solutions, strategies, and guidelines. The paper focuses on this mediation, presenting, as a case study, an adopted methodology and the first results achieved according to guidelines for the regeneration of the system of squares in the historical center of Alessandria (Piedmont, Italy). In this case, a multidisciplinary approach and a Multi-Criteria Analysis (MCA) method, supported by geospatial analysis and GIS technology, have been employed to work as mediators for a participatory process which will involve public administration, stakeholders, experts, and researchers. The paper presents an overview of the workflow, with a focus on the first set of thematic indicators and an open conclusion. It will explain how they have been defined, integrated, and turned into a dialogic tool, with the aim of laying the foundation for the next stage of involvement by the public administration and stakeholders. Specific attention will be paid to the key role of vegetational and environmental parameters, which represents the requalification strategy’s backbone, for both local and systemic scales.

Algorithm-driven activity-directed expansion of a series of antibacterial quinazolinones.

Activity-directed synthesis (ADS) is a structure-blind, function driven approach that can drive the discovery of bioactive small molecules. In ADS, arrays of reactions are designed and executed, and the crude product mixtures are then directly screened to identify reactions that yield bioactive products. The design of subsequent reaction arrays is then informed by the hit reactions that are discovered. In this study, algorithms for reaction array design were developed in which the reactions to be executed were selected from a large set of virtual reactions; the reactions were selected on the basis of similarity to reactions known to yield bioactive products. The algorithms were harnessed to design arrays of photoredox-catalysed alkylation reactions whose crude products were then screened for inhibition of growth of S. aureus ATCC29213. It was demonstrated that the approach enabled expansion of a series of antibacterial quinazolinones. It is envisaged that such algorithms could ultimately enable fully autonomous activity-directed molecular discovery.

Nanoscale Solid-Phase Isobaric Labeling for Multiplexed Quantitative Phosphoproteomics.

We established a workflow for highly sensitive multiplexed quantitative phosphoproteomics using a nanoscale solid-phase tandem mass tag (TMT) labeling reactor. Phosphopeptides were first enriched by titanium oxide chromatography and then labeled with isobaric TMT reagents in a StageTip packed with hydrophobic polymer-based sorbents. We found that TMT-labeled singly phosphorylated peptides tend to flow through the titanium oxide column. Therefore, TMT labeling should be performed after the enrichment step from tryptic peptides, resulting in the need for microscale reactions with small amounts of phosphopeptides. Using an optimized protocol for tens to hundreds of nanograms of phosphopeptides, we obtained a nearly 10-fold increase in sensitivity compared to the conventional solution-based TMT protocol. We demonstrate that this nanoscale phosphoproteomics protocol works for 50 μg of HeLa proteins treated with selumetinib, and we successfully quantified the selumetinib-regulated phosphorylated sites on a proteome scale. The MS raw data files have been deposited with the ProteomeXchange Consortium via the jPOST partner repository (https://jpostdb.org) with the data set identifier PXD025536.

Discovery of a Chiral DNA‐Targeted Platinum–Acridine Agent with Potent Enantioselective Anticancer Activity

AbstractA structure–activity relationship study was performed for a set of rigidified platinum–acridine anticancer agents containing linkers derived from chiral pyrrolidine and piperidine scaffolds. Screening a library of microscale reactions and selected resynthesized compounds in non‐small‐cell lung cancer (NSCLC) cells showed that cytotoxicities varied by more than three orders of magnitude. A potent hit compound was discovered containing a (R)‐N‐(piperidin‐3‐yl) linker (P2‐6R), which killed NCI‐H460 and A549 lung cancer cells 100 times more effectively than the S enantiomer (P2‐6S). P2‐6R accumulated in A549 cells significantly faster and produced 50‐fold higher DNA adduct levels than P2‐6S. Ligand similarity analysis suggests that only module 6R may be compatible with strainless monofunctional intercalative binding. NCI‐60 screening and COMPARE analysis highlights the spectrum of activity and potential utility of P2‐6R for treating NSCLC and other solid tumors.

Discovery of a Chiral DNA-Targeted Platinum-Acridine Agent with Potent Enantioselective Anticancer Activity.

A structure-activity relationship study was performed for a set of rigidified platinum-acridine anticancer agents containing linkers derived from chiral pyrrolidine and piperidine scaffolds. Screening a library of microscale reactions and selected resynthesized compounds in non-small-cell lung cancer (NSCLC) cells showed that cytotoxicities varied by more than three orders of magnitude. A potent hit compound was discovered containing a (R)-N-(piperidin-3-yl) linker (P2-6R), which killed NCI-H460 and A549 lung cancer cells 100 times more effectively than the S enantiomer (P2-6S). P2-6R accumulated in A549 cells significantly faster and produced 50-fold higher DNA adduct levels than P2-6S. Ligand similarity analysis suggests that only module 6R may be compatible with strainless monofunctional intercalative binding. NCI-60 screening and COMPARE analysis highlights the spectrum of activity and potential utility of P2-6R for treating NSCLC and other solid tumors.

Cover Feature: Activity‐Directed Synthesis of Inhibitors of the p53/hDM2 Protein–Protein Interaction (Chem. Eur. J. 47/2020)

Activity-directed synthesis was harnessed in the function-driven discovery of inhibitors of a protein–protein interaction. Pairs of substrates, inspired by known ligands, were exploited in 346 microscale reactions. The crude reaction mixtures were screened for biological function, and the products of promising reactions purified and characterised. Four novel and distinct inhibitor series were discovered. The artwork illustrates how motifs found in known ligands were incorporated into substrates, ultimately enabling scaffold-hopping to new inhibitor series. More information can be found in the Communication by A. Nelson et al. on page 10682.

Activity-Directed Synthesis of Inhibitors of the p53/hDM2 Protein-Protein Interaction.

Protein–protein interactions (PPIs) provide a rich source of potential targets for drug discovery and biomedical science research. However, the identification of structural‐diverse starting points for discovery of PPI inhibitors remains a significant challenge. Activity‐directed synthesis (ADS), a function‐driven discovery approach, was harnessed in the discovery of the p53/hDM2 PPI. Over two rounds of ADS, 346 microscale reactions were performed, with prioritisation on the basis of the activity of the resulting product mixtures. Four distinct and novel series of PPI inhibitors were discovered that, through biophysical characterisation, were shown to have promising ligand efficiencies. It was thus shown that ADS can facilitate ligand discovery for a target that does not have a defined small‐molecule binding site, and can provide distinctive starting points for the discovery of PPI inhibitors.

Activity-directed expansion of a series of antibacterial agents.

The feasibility of using activity-directed synthesis to drive antibacterial discovery was investigated. An array of 220 Pd-catalysed microscale reactions was executed, and the crude product mixtures were evaluated for activity against Staphylococcus aureus. Scale-up of the hit reactions, purification and evaluation, enabled expansion of a class of antibacterial quinazolinones. The novel antibacterials had MICs from 0.016 μg mL-1 (i.e. 38 nM) to 2-4 μg mL-1 against S. aureus ATCC29213.

Diagenesis of mollusc aragonite and the role of fluid reservoirs

Biogenic carbonate minerals are widely used as archives in paleoenvironmental research, providing substantial information for past depositional and diagenetic regimes. However, nearly all biogenic carbonates undergo post-mortem diagenetic alteration to variable degrees. Diagenetic features are essentially caused by complex fluid-solid interaction including recrystallization and neomorphosis of shell architecture and related geochemical resetting. A common conception is that a given primary shell texture is replaced by a secondary fabric via micro-scale dissolution-reprecipitation reactions that may reach geochemical and/or isotopic equilibrium with the diagenetic fluid. Here we document that the process of petrographic and geochemical alteration of a biogenic carbonate archive progresses stepwise, and the re-equilibration processes can be separated in space and time. More specifically, attached and bound aqueous fluids within the shell organic matter and fluid inclusions likely play a crucial role in the early stages of alteration within a relatively rock-buffered system. Degradation of organics was observed via decreased sulfur concentration and decreased fluorescence. In this early stage of alteration, the conversion of micro-scale aragonite biominerals to metastable diagenetic calcite (meso)crystals is documented by RAMAN spectroscopy, electron backscatter diffraction (EBSD), and a decrease in strontium concentrations. Further stabilization to larger, neomorphosed calcite crystals resulted from re-equilibration with external fluids and was evidenced by EBSD, oxygen isotope data, and further reduction of strontium. Spatial chemical and isotope data were used to decipher the impact of fluid availability and transfer behavior, i.e. carbonate-buffered versus diagenetic-fluid buffered, and isotope and element exchange behavior, and analyzed in context to textural alteration features. These data suggest that during diagenesis, the evolution to more sustainable (diagenetically induced) fabrics is accompanied by individual reaction steps traced by elemental and isotopic signatures. Kinetics, and thus degree of diagenetic alteration of biominerals at a given exposure to physicochemical alteration conditions is initially strongly controlled by the micro- to nano-scale internal architecture governing the availability and transfer of aqueous fluids. Results shown here have significance for those concerned with biogenic carbonate archives and have wider implications for a mechanistic understanding of carbonate diagenesis.

Droplet Incubation and Splitting in Open Microfluidic Channels.

Droplet-based microfluidics enables compartmentalization and controlled manipulation of small volumes. Open microfluidics provides increased accessibility, adaptability, and ease of manufacturing compared to closed microfluidic platforms. Here, we begin to build a toolbox for the emerging field of open channel droplet-based microfluidics, combining the ease of use associated with open microfluidic platforms with the benefits of compartmentalization afforded by droplet-based microfluidics. We develop fundamental microfluidic features to control droplets flowing in an immiscible carrier fluid within open microfluidic systems. Our systems use capillary flow to move droplets and carrier fluid through open channels and are easily fabricated through 3D printing, micromilling, or injection molding; further, droplet generation can be accomplished by simply pipetting an aqueous droplet into an empty open channel. We demonstrate on-chip incubation of multiple droplets within an open channel and subsequent transport (using an immiscible carrier phase) for downstream experimentation. We also present a method for tunable droplet splitting in open channels driven by capillary flow. Additional future applications of our toolbox for droplet manipulation in open channels include cell culture and analysis, on-chip microscale reactions, and reagent delivery.

An Isotopic Labelling Strategy to Study Cytochrome P450 Oxidations of Terpenes.

The cytochrome P450 monooxygenase CYP267B1 from Sorangium cellulosum was applied for the enzymatic oxidation of the sesquiterpene alcohols T-muurolol and isodauc-8-en-11-ol. Various isotopically labelled geranyl and farnesyl diphosphates were used for product identification from micro-scale reactions, for the determination of the absolute configurations of unknown compounds, to follow the stereochemical course of a cytochrome P450-catalysed hydroxylation step, and to investigate kinetic isotope effects. Overall, this study demonstrates that isotopically labelled terpene precursors are highly useful to follow cytochrome P450 dependent oxidations of terpenes.

Linker design for the modular assembly of multifunctional and targeted platinum(ii)-containing anticancer agents.

A versatile and efficient modular synthetic platform was developed for assembling multifunctional conjugates and targeted forms of platinum-(benz)acridines, a class of highly cytotoxic DNA-targeted hybrid agents. The synthetic strategy involved amide coupling between succinyl ester-modified platinum compounds (P1, P2) and a set of 11 biologically relevant primary and secondary amines (N1-N11). To demonstrate the feasibility and versatility of the approach, a structurally and functionally diverse range of amines was introduced. These include biologically active molecules, such as rucaparib (a PARP inhibitor), E/Z-endoxifen (an estrogen receptor antagonist), and a quinazoline-based tyrosine kinase inhibitor. Micro-scale reactions in Eppendorf tubes or on 96-well plates were used to screen for optimal coupling conditions in DMF solution with carbodiimide-, uronium-, and phosphonium-based compounds, as well as other common coupling reagents. Reactions with the phosphonium-based coupling reagent PyBOP produced the highest yields and gave the cleanest conversions. Furthermore, it was demonstrated that the chemistry can also be performed in aqueous media and is amenable to parallel synthesis based on multiple consecutive reactions in DMF in a "one-tube" format. In-line LC-MS was used to assess the stability of the conjugates in physiologically relevant buffers. Hydrolysis of the conjugates occurs at the ester moiety and is facilitated by the aquated metal moiety under low-chloride ion conditions. The rate of ester cleavage greatly depends on the nature of the amine component. Potential applications of the linker technology are discussed.

Fast and efficient microscale radiosynthesis of 3′-deoxy-3′-[18F]fluorothymidine

Microscale radiosynthesis on a miniaturized device has been reported as a promising tool for positron emission tomography. Here, we studied miscroscale-level radiosynthetic conditions by examining several microscale reactions that affect the radiochemical yield of 3′-deoxy-3′-[18F]fluorothymidine ([18F]FLT). [18F]FLT was prepared with 3-N-Boc-5′-O-dimethoxytrityl-3′-O-nosyl-thymidine using the cartridge purification method. [18F]FLT was prepared with a yield of 71%, and the product's purity was greater than 99%. Our results provide a new guideline for microscale [18F]FLT radiosynthesis.

Efficient microscale synthesis of [18F]-2-fluoro-2-deoxy-d-glucose

• [ 18 F]fluorination efficiency can be enhanced by flexible aliphatic tertiary alcohols. • [ 18 F]FDG was synthesized from mannose triflate on a micro-scale level with a higher radiochemical yield within shorter time. • Radiosynthesis of [ 18 F]FDG was influenced by ratio of tertiary alcohols and DMSO. • The concentrations of mannose triflate and bases influence on the microscale substitution reaction yield. Microscale radiosynthesis of [ 18 F]FDG using a miniaturized device has been reported as a promising tool for positron emission tomography. However, small scale [ 18 F]FDG radiosynthesis on micro fluidic devices need to be studied further to evaluate its usefulness. This paper analyzes several micro-scale reactions of [ 18 F]FDG with various reaction conditions to achieve a higher radiochemical yield of [ 18 F]FDG. Synthesis of [ 18 F]FDG was carried out using mannose triflate on a microchip, and the crude product was purified via cartridge purification. The reaction conditions were optimized by examining several factors including reaction solvents, bases and their concentrations, that influence microscale radio synthesis of [ 18 F]FDG. Based on the investigation of several variables and their effects on the radio-synthetic reaction, [ 18 F]FDG was successfully synthesized on a micro-scale level with a radiochemical yield of 71% and within shorter time frame than that of the traditional method. Moreover, purity of the product was greater than 99%. These results show that the new guideline provides a more efficient and effective method of microscale [ 18 F]FDG radiosynthesis.