Photochemical Reactions in the Synthesis of Protein–Drug Conjugates

The ability to modify biologically active molecules such as antibodies with drug molecules, fluorophores or radionuclides is crucial in drug discovery and target identification. Classic chemistry used for protein functionalisation relies almost exclusively on thermochemically mediated reactions. Our recent experiments have begun to explore the use of photochemistry to effect rapid and efficient protein functionalisation. This article introduces some of the principles and objectives of using photochemically activated reagents for protein ligation. The concept of simultaneous photoradiosynthesis of radiolabelled antibodies for use in molecular imaging is introduced as a working example. Notably, the goal of producing functionalised proteins in the absence of pre-association (non-covalent ligand-protein binding) introduces requirements that are distinct from the more regular use of photoactive groups in photoaffinity labelling. With this in mind, the chemistry of thirteen different classes of photoactivatable reagents that react through the formation of intermediate carbenes, electrophiles, dienes, or radicals, is assessed.     

Effect of Headgroups on Small‐Ion Permeability across Archaea‐Inspired Tetraether Lipid Membranes

This paper examines the effects of four different polar headgroups on small‐ion membrane permeability from liposomes comprised of Archaea‐inspired glycerolmonoalkyl glycerol tetraether (GMGT) lipids. We found that the membrane‐leakage rate across GMGT lipid membranes varied by a factor of ≤1.6 as a function of headgroup structure. However, the leakage rates of small ions across membranes comprised of commercial bilayer‐forming 1‐palmitoyl‐2‐oleoyl‐sn‐glycerol (PO) lipids varied by as much as 32‐fold within the same series of headgroups. These results demonstrate that membrane leakage from GMGT lipids is less influenced by headgroup structure, making it possible to tailor the structure of the polar headgroups on GMGT lipids while retaining predictable leakage properties of membranes comprised of these tethered lipids.     

Tuning Tetrazole Photochemistry for Protein Ligation and Molecular Imaging

Photochemistry provides a wide range of alternative reagents that hold potential for use in bimolecular functionalisation of proteins. Here, we report the synthesis and characterisation of metal ion binding chelates derivatised with disubstituted tetrazoles for the photoradiochemical labelling of monoclonal antibodies (mAbs). The photophysical properties of tetrazoles featuring extended aromatic systems and auxochromic substituents to tune excitation toward longer wavelengths (365 and 395 nm) were studied. Two photoactivatable chelates based on desferrioxamine B (DFO) and the aza-macrocycle NODAGA were functionalised with a tetrazole and developed for protein labelling with ⁸⁹Zr, ⁶⁴Cu and ⁶⁸Ga radionuclides. DFO-tetrazole ( 1 ) was assessed by direct conjugation to formulated trastuzumab and subsequent radiolabelling with ⁸⁹Zr. Radiochemical studies and cellular-based binding assays demonstrated that the radiotracer remained stable in vitro retained high immunoreactivity. Positron emission tomography (PET) imaging and biodistribution studies were used to measure the tumour specific uptake and pharmacokinetic profile in mice bearing SK-OV-3 xenografts. Experiments demonstrate that tetrazole-based photochemistry is a viable approach for the light-induced synthesis of PET radiotracers.     

Dynamical versus variational symmetries: understanding Noether's first theorem

It is argued that awareness of the distinction between dynamical and variational symmetries is crucial to understanding the significance of Noether's 1918 work. Special attention is paid, by way of a number of striking examples, to Noether's first theorem, which establishes a correlation between dynamical symmetries and conservation principles.     

An optimized method for the synthesis of 2,6-diaminoanthracene

The reduction of 2,6-diaminoanthraquinone to 2,6-diaminoanthracene was examined under a variety of conditions. Direct reduction using zinc powder gave a mixture of the target product and 9,10-dihydro-2,6-diaminoanthracene under all the conditions examined. Protection of the starting amine, followed by borohydride reduction and deprotection, gave the target product in 14-50% yield. Finally, tin powder was used to reduce the anthraquinone to 2,6-diaminoanthrone in quantitative yield. This compound was further reduced to the target 2,6-diaminoanthracene in 55-65% yield.     

The importance of chain length for the polyphosphate enhancement of acidic potassium permanganate chemiluminescence

Sodium polyphosphate is commonly used to enhance chemiluminescence reactions with acidic potassium permanganate through a dual enhancement mechanism, but commercially available polyphosphates vary greatly in composition. We have examined the influence of polyphosphate composition and concentration on both the dual enhancement mechanism of chemiluminescence intensity and the stability of the reagent under analytically useful conditions. The average chain length (n) provides a convenient characterisation, but materials with similar values can exhibit markedly different distributions of phosphate oligomers. There is a minimum polyphosphate chain length (∼6) required for a large enhancement of the emission intensity, but no further advantage was obtained using polyphosphate materials with much longer average chain lengths. Providing there is a sufficient average chain length, the optimum concentration of polyphosphate is dependent on the analyte and in some cases, may be lower than the quantities previously used in routine detection. However, the concentration of polyphosphate should not be lowered in permanganate reagents that have been partially reduced to form high concentrations of the key manganese(III) co-reactant, as this intermediate needs to be stabilised to prevent formation of insoluble manganese(IV).     

Thermographic signal reconstruction for vibrothermography

Vibrothermography, also known as thermosonics or sonic infrared, is a method of nondestructive evaluation that finds cracks or delaminations from the heat given off in response to vibration. In vibrothermography, finding cracks requires identifying and localizing pulsed surface and subsurface heat sources from a time sequence of infrared images. Traditionally this identification involves manually stepping through and studying the images. Careful observation of the heating and subsequent cooling is needed to distinguish cracks from false indications. In this paper, we present an algorithm that reduces the entire time sequence to a single static plot. The plot uses only a few coefficients per pixel to reconstruct the original sequence; this is possible because the reduction is based on a physical model. As an added bonus, the algorithm reduces noise and improves sensitivity. A single false-color image summarizes all the information from the entire sequence, simplifying the task of identifying cracks.     

Multi-scale magnetic resonance measurements and validation of Discrete Element Model simulations

This short review describes the capabilities of magnetic resonance (MR) to image opaque single- and two-phase granular systems, such as rotating cylinders and gas-fluidized beds operated in different fluidization regimes. The unique capability of MR to not only image the solids’ distribution (voidage) but also the velocity of the particulate phase is clearly shown. It is demonstrated that MR can provide measurements over different length and time scales. With the MR equipment used for the studies summarized here, temporal and spatial scales range from sub-millisecond to hours and from a few hundred micrometres to a few centimetres, respectively. Besides providing crucial data required for an improved understanding of the underlying physics of granular flows, multi-scale MR measurements were also used to validate numerical simulations of granular systems. It is shown that predictions of time-averaged properties, such as voidage and velocity of the particulate phase, made using the Discrete Element Model agree very well with MR measurements.     

Mass Activated Droplet Sorting (MADS) Enables High‐Throughput Screening of Enzymatic Reactions at Nanoliter Scale

Microfluidic droplet sorting enables the high‐throughput screening and selection of water‐in‐oil microreactors at speeds and volumes unparalleled by traditional well‐plate approaches. Most such systems sort using fluorescent reporters on modified substrates or reactions that are rarely industrially relevant. We describe a microfluidic system for high‐throughput sorting of nanoliter droplets based on direct detection using electrospray ionization mass spectrometry (ESI‐MS). Droplets are split, one portion is analyzed by ESI‐MS, and the second portion is sorted based on the MS result. Throughput of 0.7 samples s^?1 is achieved with 98 % accuracy using a self‐correcting and adaptive sorting algorithm. We use the system to screen ≈15 000 samples in 6 h and demonstrate its utility by sorting 25 nL droplets containing transaminase expressed in vitro. Label‐free ESI‐MS droplet screening expands the toolbox for droplet detection and recovery, improving the applicability of droplet sorting to protein engineering, drug discovery, and diagnostic workflows.