Formation of left-handed helices by C2'-fluorinated nucleic acids under physiological salt conditions.

Recent findings in cell biology have rekindled interest in Z-DNA, the left-handed helical form of DNA. We report here that two minimally modified nucleosides, 2'F-araC and 2'F-riboG, induce the formation of the Z-form under low ionic strength. We show that oligomers entirely made of these two nucleosides exclusively produce left-handed duplexes that bind to the Zα domain of ADAR1. The effect of the two nucleotides is so dramatic that Z-form duplexes are the only species observed in 10 mM sodium phosphate buffer and neutral pH, and no B-form is observed at any temperature. Hence, in contrast to other studies reporting formation of Z/B-form equilibria by a preference for purine glycosidic angles in syn, our NMR and computational work revealed that sequential 2'F…H2N and intramolecular 3'H…N3' interactions stabilize the left-handed helix. The equilibrium between B- and Z- forms is slow in the 19F NMR time scale (≥ms), and each conformation exhibited unprecedented chemical shift differences in the 19F signals. This observation led to a reliable estimation of the relative population of B and Z species and enabled us to monitor B-Z transitions under different conditions. The unique features of 2'F-modified DNA should thus be a valuable addition to existing techniques for specific detection of new Z-binding proteins and ligands.

Factorized Quadruples and a Predictor of Higher-Level Correlation in Thermochemistry.

Coupled cluster theory has had a momentous impact on the ab initio prediction of molecular properties, and remains a staple ingratiate in high-accuracy thermochemical model chemistries. However, these methods require inclusion of at least some connected quadruple excitations, which generally scale at best as O(N9) with the number of basis functions. It is very difficult to predict, a priori, the effect correlation of past CCSD(T) on a given reaction energy. The purpose of this work is to examine cost-effective quadruple corrections based on the factorization theorem of the many-body perturbation theory that may address these challenges. We show that the O(N7) factorized CCSD(TQf) method introduces minimal error to predicted correlation and reaction energies as compared to the O(N9) CCSD(TQ). Further, we examine the performance of Goodson's continued fraction method in the estimation of CCSDT(Q)Λ contributions to reaction energies as well as a "new" method related to %TAE[(T)] that we refer to as a scaled perturbation estimator. We find that the scaled perturbation estimator based upon CCSD(TQf)/cc-pVDZ is capable of predicting CCSDT(Q)Λ/cc-pVDZ contributions to reaction energies with an average error of 0.07 kcal mol-1 and an L2D of 0.52 kcal mol-1 when applied to a test-suite of nearly 3000 reactions. This offers a means by which to reliably "ballpark" how important post-CCSD(T) contributions are to reaction energies while incurring no more than CCSD(T) formal cost and a little mental math.

Sub 20 cm-1 computational prediction of the CH bond energy - a case of systematic error in computational thermochemistry.

The bond dissociation energy of methylidyne, D0(CH), is studied using an improved version of the High-Accuracy Extrapolated ab initio Thermochemistry (HEAT) approach as well as the Feller-Peterson-Dixon (FPD) model chemistry. These calculations, which include basis sets up to nonuple (aug-cc-pCV9Z) quality, are expected to be capable of providing results substantially more accurate than the ca. 1 kJ mol-1 level that is characteristic of standard high-accuracy protocols for computational thermochemistry. The calculated 0 K CH bond energy (27 954 ± 15 cm-1 for HEAT and 27 956 ± 15 cm-1 for FPD), along with equivalent treatments of the CH ionization energy and the CH+ dissociation energy (85 829 ± 15 cm-1 and 32 946 ± 15 cm-1, respectively), were compared to the existing benchmarks from Active Thermochemical Tables (ATcT), uncovering an unexpected difference for D0(CH). This has prompted a detailed reexamination of the provenance of the corresponding ATcT benchmark, allowing the discovery and subsequent correction of a systematic error present in several published high-level calculations, ultimately yielding an amended ATcT benchmark for D0(CH). Finally, the current theoretical results were added to the ATcT Thermochemical Network, producing refined ATcT estimates of 27 957.3 ± 6.0 cm-1 for D0(CH), 32 946.7 ± 0.6 cm-1 for D0(CH+), and 85 831.0 ± 6.0 cm-1 for IE(CH).

Recent progress in non-native nucleic acid modifications.

While Nature harnesses RNA and DNA to store, read and write genetic information, the inherent programmability, synthetic accessibility and wide functionality of these nucleic acids make them attractive tools for use in a vast array of applications. In medicine, antisense oligonucleotides (ASOs), siRNAs, and therapeutic aptamers are explored as potent targeted treatment and diagnostic modalities, while in the technological field oligonucleotides have found use in new materials, catalysis, and data storage. The use of natural oligonucleotides limits the possible chemical functionality of resulting technologies while inherent shortcomings, such as susceptibility to nuclease degradation, provide obstacles to their application. Modified oligonucleotides, at the level of the nucleobase, sugar and/or phosphate backbone, are widely used to overcome these limitations. This review provides the reader with an overview of non-native modifications and the challenges faced in the design, synthesis, application and outlook of novel modified oligonucleotides.

Elaborated thermochemical treatment of HF, CO, N2, and H2O: Insight into HEAT and its extensions.

Empirical, highly accurate non-relativistic electronic total atomization energies (eTAEs) are established by combining experimental or computationally converged treatments of the nuclear motion and relativistic contributions with the total atomization energies of HF, CO, N2, and H2O obtained from the Active Thermochemical Tables. These eTAEs, which have estimated (2σ) uncertainties of less than 10 cm-1 (0.12 kJ mol-1), form the basis for an analysis of high-level ab initio quantum chemical calculations that aim at reproducing these eTAEs for the title molecules. The results are then employed to analyze the performance of the high-accuracy extrapolated ab initio thermochemistry, or High-Accuracy Extrapolated Ab Initio Thermochemistry (HEAT), family of theoretical methods. The method known as HEAT-345(Q), in particular, is found to benefit from fortuitous error cancellation between its treatment of the zero-point energy, extrapolation errors in the Hartree-Fock and coupled cluster contributions, neglect of post-(T) core-correlation, and the basis-set error involved in higher-level correlation corrections. In addition to shedding light on a longstanding curiosity of the HEAT protocol-where the cheapest HEAT-345(Q) performs comparably to the theoretically more complete HEAT-456QP procedure-this study lays the foundation for extended HEAT variants that offer substantial improvements in accuracy relative to the established approaches.

Using isotopologues to probe the potential energy surface of reactions of C2H2 ++C3H4.

Investigations into bimolecular reaction kinetics probe the details of the underlying potential energy surface (PES), which can help to validate high-level quantum chemical calculations. We utilize a combined linear Paul ion trap with a time-of-flight mass spectrometer to study isotopologue reactions between acetylene cations (C2H2 +) and two isomers of C3H4: propyne (HC3H3) and allene (H2C3H2). In a previous study [Schmid et al., Phys. Chem. Chem. Phys. 22, 20303 (2020)],1 we showed that the two isomers of C3H4 have fundamentally different reaction mechanisms. Here, we further explore the calculated PES by isotope substitution. While isotopic substitution of reactants is a standard experimental tool in the investigation of molecular reaction kinetics, the controlled environment of co-trapped, laser-cooled Ca+ ions allows the different isotopic reaction pathways to be followed in greater detail. We report branching ratios for all of the primary products of the different isotopic species. The results validate the previously proposed mechanism: propyne forms a bound reaction complex with C2H2 +, while allene and C2H2 + perform long-range charge exchange only.

Mechanochemical Synthesis of Short DNA Fragments.

We demonstrate the first mechanochemical synthesis of DNA fragments by ball milling, enabling the synthesis of oligomers of controllable sequence and length using multi-step, one-pot reactions, without bulk solvent or the need to isolate intermediates. Mechanochemistry allowed for coupling of phosphoramidite monomers to the 5'-hydroxyl group of nucleosides, iodine/water oxidation of the resulting phosphite triester linkage, and removal of the 5'-dimethoxytrityl (DMTr) protecting group in situ in good yields (up to 60 % over three steps) to produce DNA dimers in a one-pot manner. H-Phosphonate chemistry under milling conditions enabled coupling and protection of the H-phosphonate linkage, as well as removal of the 5'-DMTr protecting group in situ, enabling a one-pot process with good yields (up to 65 % over three steps, or ca. 87 % per step). Sulfurization of the internucleotide linkage was possible using elemental sulfur (S8) or sulfur transfer reagents, yielding the target DNA phosphorothioate dimers in good yield (up to 80 % over two steps). This work opens the door to creation of solvent-free synthesis methodologies for DNA and RNA therapeutics.

Structure-based optimisation of orally active & reversible MetAP-2 inhibitors maintaining a tight 'molecular budget'.

Structure-based led optimisation of orally active reversible Methionine Aminopeptidase-2 (MetAP-2) inhibitors utilising a 'molecular budget' medicinal chemistry strategy is described. The key physicochemical parameters of target molecules (cLogP, molecular size and H-bond donor count) were monitored through straightforward and intuitive use of atom count and distribution. The balance between structure-based design and an awareness of the physicochemical properties of the compounds synthesised enabled the rapid identification of a potent molecule with good oral pharmacokinetic (PK) characteristics by making fewer, higher quality compounds. The resulting candidate quality molecule was validated in a mechanistic cellular assay and a rodent secondary immunisation model.

Exhaustive Product Analysis of Three Benzene Discharges by Microwave Spectroscopy.

Using chirped and cavity microwave spectroscopies, automated double resonance, new high-speed fitting and deep learning algorithms, and large databases of computed structures, the discharge products of benzene alone, or in combination with molecular oxygen or nitrogen, have been exhaustively characterized between 6.5 and 26 GHz. In total, more than 3300 spectral features were observed; 89% of these, accounting for 97% of the total intensity, have now been assigned to 152 distinct chemical species and 60 of their variants (i.e., isotopic species and vibrationally excited states). Roughly 50 of the products are entirely new or poorly characterized at high resolution, including many heavier by mass than the precursor benzene. These findings provide direct evidence for a rich architecture of two- and three-dimensional carbon and indicate that benzene growth, particularly the formation of ring-chain molecules, occurs facilely under our experimental conditions. The present analysis also illustrates the utility of microwave spectroscopy as a precision tool for complex mixture analysis, irrespective of whether the rotational spectrum of a product species is known a priori or not. From this large quantity of data, for example, it is possible to determine with confidence the relative abundances of different product masses, but more importantly the relative abundances of different isomers with the same mass. The complementary nature of this type of analysis to traditional mass spectrometry is discussed.

Structure guided drug design to develop kallikrein 5 inhibitors to treat Netherton syndrome.

The connection between Netherton syndrome and overactivation of epidermal/dermal proteases particularly KLK5 has been well established. To treat sufferers of this severe condition we wished to develop a topical KLK5 inhibitor in order to normalise epidermal shedding and reduce the associated inflammation and itching. In this paper we describe structure-based optimisation of a series of brightly coloured weak KLK5 inhibitors into colourless, non-irritant molecules with good KLK5 activity and selectivity over a range of serine proteases.

Kallikrein 5 inhibitors identified through structure based drug design in search for a treatment for Netherton Syndrome.

Netherton syndrome (NS) is a rare and debilitating severe autosomal recessive genetic skin disease with high mortality rates particularly in neonates. NS is caused by loss-of-function SPINK5 mutations leading to unregulated kallikrein 5 (KLK5) and kallikrein 7 (KLK7) activity. Furthermore, KLK5 inhibition has been proposed as a potential therapeutic treatment for NS. Identification of potent and selective KLK5 inhibitors would enable further exploration of the disease biology and could ultimately lead to a treatment for NS. This publication describes how fragmentation of known trypsin-like serine protease (TLSP) inhibitors resulted in the identification of a series of phenolic amidine-based KLK5 inhibitors 1. X-ray crystallography was used to find alternatives to the phenol interaction leading to identification of carbonyl analogues such as lactam 13 and benzimidazole 15. These reversible inhibitors, with selectivity over KLK1 (10-100 fold), provided novel starting points for the guided growth towards suitable tool molecules for the exploration of KLK5 biology.

Design and development of a series of borocycles as selective, covalent kallikrein 5 inhibitors.

The connection between Netherton syndrome and overactivation of epidermal/dermal proteases, particularly Kallikrein 5 (KLK5) has been well established and it is expected that a KLK5 inhibitor would improve the dermal barrier and also reduce the pain and itch that afflict Netherton syndrome patients. One of the challenges of covalent protease inhibitors has been achieving selectivity over closely related targets. In this paper we describe the use of structural insight to design and develop a selective and highly potent reversibly covalent KLK5 inhibitor from an initial weakly binding fragment.

High-accuracy extrapolated ab initio thermochemistry. IV. A modified recipe for computational efficiency.

A number of economical modifications to the high-accuracy extrapolated ab initio thermochemistry (HEAT) model chemistry are evaluated. The two resulting schemes, designated as mHEAT and mHEAT+, are designed for efficient and pragmatic evaluation of molecular energies in systems somewhat larger than can be practically studied by the unapproximated HEAT scheme. It is found that mHEAT+ produces heats of formation with nearly subchemical (±1 kJ/mol) accuracy at a substantially reduced cost relative to the full scheme. Total atomization energies calculated using the new thermochemical recipes are compared to the results of the HEAT-345(Q) model chemistry, and enthalpies of formation for the three protocols are also compared to Active Thermochemical Tables. Finally, a small selection of transition states is studied using mHEAT and mHEAT+, which illuminates some interesting features of reaction barriers and serves as an initial benchmark of the performance of these model chemistries for chemical kinetics applications.

Thermal Decomposition of Potential Ester Biofuels. Part I: Methyl Acetate and Methyl Butanoate.

Two methyl esters were examined as models for the pyrolysis of biofuels. Dilute samples (0.06-0.13%) of methyl acetate (CH3COOCH3) and methyl butanoate (CH3CH2CH2COOCH3) were entrained in (He, Ar) carrier gas and decomposed in a set of flash-pyrolysis microreactors. The pyrolysis products resulting from the methyl esters were detected and identified by vacuum ultraviolet photoionization mass spectrometry. Complementary product identification was provided by matrix infrared absorption spectroscopy. Pyrolysis pressures in the pulsed microreactor were about 20 Torr and residence times through the reactors were roughly 25-150 µs. Reactor temperatures of 300-1600 K were explored. Decomposition of CH3COOCH3 commences at 1000 K, and the initial products are (CH2═C═O and CH3OH). As the microreactor is heated to 1300 K, a mixture of CH2═C═O and CH3OH, CH3, CH2═O, H, CO, and CO2 appears. The thermal cracking of CH3CH2CH2COOCH3 begins at 800 K with the formation of CH3CH2CH═C═O and CH3OH. By 1300 K, the pyrolysis of methyl butanoate yields a complex mixture of CH3CH2CH═C═O, CH3OH, CH3, CH2═O, CO, CO2, CH3CH═CH2, CH2CHCH2, CH2═C═CH2, HCCCH2, CH2═C═C═O, CH2═CH2, HC≡CH, and CH2═C═O. On the basis of the results from the thermal cracking of methyl acetate and methyl butanoate, we predict several important decomposition channels for the pyrolysis of fatty acid methyl esters, R-CH2-COOCH3. The lowest-energy fragmentation will be a 4-center elimination of methanol to form the ketene RCH═C═O. At higher temperatures, concerted fragmentation to radicals will ensue to produce a mixture of species: (RCH2 + CO2 + CH3) and (RCH2 + CO + CH2═O + H). Thermal cracking of the ß C-C bond of the methyl ester will generate the radicals (R and H) as well as CH2═C═O + CH2═O. The thermochemistry of methyl acetate and its fragmentation products were obtained via the Active Thermochemical Tables (ATcT) approach, resulting in ΔfH298(CH3COOCH3) = -98.7 ± 0.2 kcal mol-1, ΔfH298(CH3CO2) = -45.7 ± 0.3 kcal mol-1, and ΔfH298(COOCH3) = -38.3 ± 0.4 kcal mol-1.

Electron Transfer Facilitated by Dendrimer-Encapsulated Pt Nanoparticles Across Ultrathin, Insulating Oxide Films.

Electrocatalytic oxygen reduction at carbon electrodes fully passivated by Al2O3 is reported. Specifically, pyrolyzed polymer film (PPF) electrodes were prepared and then coated with pinhole-free Al2O3 layers ranging in thickness from 2.5 to 5.7 nm. All of these ultrathin oxide film thicknesses completely passivated the PPF electrodes, resulting in no faradaic current for either inner-sphere or outer-sphere electrochemical reactions. The electrodes could, however, be reactivated by immobilizing Pt dendrimer-encapsulated nanoparticles (DENs), containing an average of 55 atoms each, on the oxide surface. These PPF/Al2O3/Pt DEN electrodes were completely stable under a variety of electrochemical and solution conditions, and they are active for simple electron-transfer reactions and for more complex electrocatalytic processes. This approach for preparing well-defined oxide electrodes opens the door to a better understanding of the effect of oxide supports on reactions electrocatalyzed by metal nanoparticles.

A Potent and Selective Kallikrein-5 Inhibitor Delivers High Pharmacological Activity in Skin from Patients with Netherton Syndrome.

Regulation of proteolytic activity in the skin plays a pivotal role in epidermal homeostasis. This is best exemplified in Netherton syndrome, a severe genetic skin condition caused by loss-of-function mutations in the gene serine protease inhibitor Kazal-type 5 encoding lympho-epithelial Kazal-type-related inhibitor, a serine protease inhibitor that regulates kallikrein (KLK)-related peptidase 5, 7, and 14 activities. KLK5 plays a central role in stratum corneum shedding and inflammatory cell signaling, activates KLK7 and KLK14, and is therefore an optimal therapeutic target. We aimed to identify a potent and selective small-molecule inhibitor of KLK5 amenable to epidermal delivery. GSK951 was identified using a structure-based design strategy and showed a half maximal inhibitory concentration of 250 pM for KLK5 and greater than 100-fold selectivity over KLK7 and KLK14. Cocrystal structure analysis identified the critical catalytic site interactions to a surrogate for KLK5. Topical application of GSK951-containing cream inhibited KLK5 activity in TgKLK5 mouse skin, reduced transepidermal water loss, and decreased proinflammatory cytokine expression. GSK951 achieved high concentrations in healthy human epidermis following topical application in a cream formulation. Finally, KLK5 protease activity was increased in stratum corneum of patients with Netherton syndrome and significantly inhibited by GSK951. These findings unveil a KLK5-specific small-molecule inhibitor with a high therapeutic potential for patients with Netherton syndrome.

Tackling burnout: why is it important?

Evidence suggests a significant relationship between healthcare staff wellbeing and patient safety, with burnout directly and indirectly affecting medicolegal risk. Poor wellbeing of doctors has major implications for patient outcomes and the overall performance of healthcare organisations. This editorial looks at the predisposing factors that can lead to burnout and the potential solutions.

Cocktailed fragment screening by X-ray crystallography of the antibacterial target undecaprenyl pyrophosphate synthase from Acinetobacter baumannii.

Direct soaking of protein crystals with small-molecule fragments grouped into complementary clusters is a useful technique when assessing the potential of a new crystal system to support structure-guided drug discovery. It provides a robustness check prior to any extensive crystal screening, a double check for assay binding cutoffs and structural data for binding pockets that may or may not be picked out in assay measurements. The structural output from this technique for three novel fragment molecules identified to bind to the antibacterial target Acinetobacter baumannii undecaprenyl pyrophosphate synthase are reported, and the different physicochemical requirements of a successful antibiotic are compared with traditional medicines.

Characterising the role of small airways in severe asthma using low frequency forced oscillations: A combined computational and clinical approach.

BACKGROUND: Within asthma, the small airways (≤2 mm in diameter) play an important role in pathophysiology. Using a combined clinical-computational approach, we sought to more precisely evaluate the contribution of the small airways to deep-breath induced airway dilation (in the absence of bronchial challenge), which may be impaired in severe asthma. METHODS: A patient-based computational model of the FOT was used to examine the sensitivity and specificity of FOT signals to small airways constriction at frequencies of 2 & 8 Hz. A clinical study of moderate to severe asthmatics (n = 24), and healthy volunteers (n = 10) was performed to evaluate correlations between baseline and post deep inspiration (following bronchodilator withhold and in the absence of prior bronchial challenge) forced oscillation technique (FOT) responses (at 2Hz and 8Hz) and asthma treatment intensity, spirometry, airway hyper-responsiveness and airway inflammation. RESULTS: Computational modelling demonstrated that baseline resistance measures at 2Hz are both sensitive and specific to anatomical narrowing in the small airways. Furthermore, small airways resistance was significantly increased in asthmatics compared to health. Despite these differences, there were no noticeable differences between asthmatics and healthy volunteers in resistive measures following deep inspiration (DI) and DI responses of small airways were amplified in the presence of spirometry defined airflow limitation. CONCLUSIONS: These results suggest that the small airways demonstrate increased resistance in moderate-to-severe asthma but dilate normally in response to deep inspirations in the absence of bronchial challenge. This suggests that effective targeting of the small airways is required to achieve functional improvements in moderate-severe asthmatic patients.