Halomethyl-Triazoles for Rapid, Site-Selective Protein Modification

Post-translational modifications (PTMs) are used by organisms to control protein structure and function after protein translation, but their study is complicated and their roles are not often well understood as PTMs are difficult to introduce onto proteins selectively. Designing reagents that are both good mimics of PTMs, but also only modify select amino acid residues in proteins is challenging. Frequently, both a chemical warhead and linker are used, creating a product that is a misrepresentation of the natural modification. We have previously shown that biotin-chloromethyl-triazole is an effective reagent for cysteine modification to give S-Lys derivatives where the triazole is a good mimic of natural lysine acylation. Here, we demonstrate both how the reactivity of the alkylating reagents can be increased and how the range of triazole PTM mimics can be expanded. These new iodomethyl-triazole reagents are able to modify a cysteine residue on a histone protein with excellent selectivity in 30 min to give PTM mimics of acylated lysine side-chains. Studies on the more complicated, folded protein SCP-2L showed promising reactivity, but also suggested the halomethyl-triazoles are potent alkylators of methionine residues

New methods for the synthesis of spirocyclic cephalosporin analogues

Spiro compounds provide attractive targets in drug discovery due to their inherent three-dimensional structures, which enhance protein interactions, aid solubility and facilitate molecular modelling. However, synthetic methodology for the spiro-functionalisation of important classes of penicillin and cephalosporin β-lactam antibiotics is comparatively limited. We report a novel method for the generation of spiro-cephalosporin compounds through a Michael-type addition to the dihydrothiazine ring. Coupling of a range of catechols is achieved under mildly basic conditions (K2CO3, DMF), giving the stereoselective formation of spiro-cephalosporins (d.r. 14:1 to 8:1) in moderate to good yields (28−65%)

The chemical characterisation by HPLC–PDA and HPLC–ESI–MS of unaged and aged fibre samples dyed with sawwort (Serratula tinctoria L.)

The acid-hydrolysed extracts of freshly dyed reference fibres of sawwort harvested from several different geographical locations were characterised by the use of high-performance liquid chromatography with photodiode array detection (HPLC–PDA) and coupled with electrospray ionisation mass spectrometric analysis (HPLC–ESI–MS). A related species, Serratula coronata L. was also characterised. The flavonols quercetin, 3-O-methylquercetin and kaempferol, and the flavones luteolin and apigenin were observed in all samples. Accelerated ageing studies confirmed the sensitivity of the flavonol components to photo-oxidative degradation. The poor lightfastness and small relative proportion of these flavonol components found in the extracts of freshly dyed sawwort limits their use as sawwort ‘markers’ in historical samples

Studies towards the total synthesis of Disorazole C1 and its analogues

Structure–activity relationships (SARs) in the disorazole family have been revealed through the biological testing of natural disorazoles and their synthetic analogues, but little is known about the contribution of the oxazole to the anti-tubulin activity of disorazole C1 I. The development of a novel Evans–Tishchenko/alkyne metathesis (ET–AM) route towards the synthesis of disorazole C1 will provide straightforward access to disorazole C1 and its heterocyclic analogues, thus allowing the contribution of the oxazole to the natural product’s bioactivity to be elucidated. Our ET–AM approach offers a highly diastereoselective and convergent means of constructing heterocyclic analogues of the disorazole C1 scaffold Het-II. It is envisaged that ET coupling of C(1)–C(9) aldehydes Het-IV to the C(10)–C(19) β-hydroxyketone V will give the key, requisite, 1,3-anti diol monoester bis-alkynes Het-III for dimerisation via an alkyne cross-metathesis/ring-closing alkyne metathesis (ACM–RCAM) reaction. Further diversification may be achieved through the synthesis of C(6)-heteroatom analogues of the C(1)–C(9) fragment Het-IV. Chapter 2 outlines efforts towards the synthesis of C(6)-amino analogues Het-VI of the C(1)–C(9) fragment IV. Elaboration of Garner’s aldehyde VIII allowed the synthesis of the N-protected C(5)–C(9) mesylate VII; an analogue of an advanced C(1)–C(9) fragment intermediate. A scalable route towards the synthesis of the C(10)–C(19) fragment V and investigations into its reactivity under ET coupling conditions are critical to the success of our ET–AM approach. Chapter 3 details convergent approaches towards the synthesis of the C(10)–C(19) β-hydroxyketone V, which centred around: (i) an olefin cross-metathesis reaction [C(11)–C(12) disconnection]; (ii) an epoxide ringopening reaction [C(12)–C(13) disconnection]; and (iii) a Mukaiyama aldol reaction [C(14)–C(15) disconnection]. Chapter 4 describes our successful linear synthesis of the β-hydroxyketone V. Gram-scale preparation of the C(10)–C(19) fragment V permitted investigation into the viability of the ET reaction as a fragment coupling strategy, the results of which are reported in Chapter 5. Although many (hetero)aryl aldehydes failed to react, the successful coupling of electron-deficient substrates allowed a contingency strategy to be explored through preparation of the mono-protected diol IX. Esterification of IX with the carboxylic acid derivative of the C(1)–C(9) oxazole has allowed generation of the C(1)–C(9)/C(10')–C(19') bis-alkyne X required for future AM investigations

An optimised small-scale sample preparation workflow for historical dye analysis using UHPLC-PDA applied to Scottish and English Renaissance embroidery

A sample preparation workflow for historical dye analysis based on 96 well plates and filtration by centrifugation was developed. It requires less sample and the introduced error is decreased, making it useful for culturally important objects. A sample preparation workflow for historical dye analysis requiring less sample has been developed. Samples as small as 0.01 ± 0.005 mg have been successfully analysed and high percentage recoveries (>85%), more automation and shorter preparation time have been achieved using filtration by centrifugation and only one manual transfer. The optimised workflow based on 96 well plates together with the shorter UHPLC method developed makes dye analysis data collection faster from unprocessed sample to result, facilitating the creation of larger datasets and application of chemometric approaches. The method was evaluated on 85 samples from 12 dye sources (RSD < 5.1%, = 5) as well as 22 samples from a 17 century embroidered stomacher from the National Museums Scotland (NMS) collection

Imaging Drug Uptake by Bioorthogonal Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy in tandem with bioorthogonal Raman labelling strategies is set to revolutionise the direct visualisation of intracellular drug uptake. Rational evaluation of a series of Raman-active labels has allowed the identification of highly active labels which have minimal perturbation on the biological efficacy of the parent drug. Drug uptake has been correlated with markers of cellular composition and cell cycle status, and mapped across intracellular structures using dual-colour and multi-modal imaging. The minimal phototoxicity and low photobleaching associated with SRS microscopy has enabled real-time imaging in live cells. These studies demonstrate the potential for SRS microscopy in the drug development process

Kinetic analysis of bioorthogonal reaction mechanisms using Raman microscopy

Raman spectroscopy is well-suited to the study of bioorthogonal reaction processes because it is a non-destructive technique, which employs relatively low energy laser irradiation, and water is only very weakly scattered in the Raman spectrum enabling live cell imaging. In addition, Raman spectroscopy allows species-specific label-free visualisation; chemical contrast may be achieved when imaging a cell in its native environment without fixatives or stains. Combined with the rapid advances in the field of Raman imaging over the last decade, particularly in stimulated Raman spectroscopy (SRS), this technique has the potential to revolutionise our mechanistic understanding of the biochemical and medicinal chemistry applications of bioorthogonal reactions. Current approaches to the kinetic analysis of bioorthogonal reactions (including heat flow calorimetry, UV-vis spectroscopy, fluorescence, IR, NMR and MS) have a number of practical shortcomings for intracellular applications. We highlight the advantages offered by Raman microscopy for reaction analysis in the context of both established and emerging bioorthogonal reactions, including the copper(i) catalysed azide-alkyne cycloaddition (CuAAC) click reaction and Glaser-Hay coupling