Loading Determination of DMTr-substituted Resins for Large-scale Oligonucleotide Synthesis.

The loading (i.e., substitution) of solid supports for oligonucleotide synthesis is an important parameter in large-scale manufacturing of oligonucleotides. Several key process parameters are dependent on the substitution of the solid support, including the number of phosphoramidite nucleoside equivalents used in the coupling step. For dimethoxytrityl (DMTr)-loaded solid supports, the substitution of the resin is determined by quantitatively cleaving the DMTr protecting group from the resin under acidic conditions and then analyzing the DMTr cation extinction by UV/vis spectroscopy. The spectrometric measurement can be performed at 409 nm or the global extinction maximum of 510 nm. The substitution is then calculated based on the Lambert-Beer law analogously to the substitution determination of Fmoc-substituted resins. Below, the determination of the molar extinction coefficient at 510 nm in a solution of 10% dichloroacetic acid in toluene and subsequent determination of the DMTr loading of DMTr-substituted resins is reported. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Determination of the molar extinction coefficient at 510 nm in DCA Deblock solution Basic Protocol 2: Substitution determination of DMTr-substituted resins by cleavage of the DMTr cation.

A conceptual framework to support hospitals to measure and realise financial benefits from process improvement programs: perspectives from Australia, USA and UK.

Objective The objective of this research is to appraise current practice in hospitals against the 'Framework to achieve value in healthcare' (the Framework) and to identify additional contributory factors that support or hinder its application. Methods A multi-site case study was undertaken with five hospitals in Australia, the USA and UK using purposeful sampling to identify hospitals to participate. Data collection took place between September and November 2022. The hospitals included in the study had Process Improvement (PI) programs of more than 5 years duration, with strong executive engagement and broad outcomes measurement, including financial benefits. All hospitals were acute public hospitals or private, not for profit. Results All hospitals indicated current practice according to Steps 1-5 for some part of their PI programs. All hospitals indicated that they were more likely to include financial benefits measurement for activities aimed specifically at improving cost rather than reducing non-value adding activities or improving the value of clinical care. Step 5 (reinvestment of cost savings) of the Framework is dependent on the accomplishment of Step 4 (measurement and realisation of financial benefits) and the contributory elements are important in supporting hospitals to utilise the Framework. Conclusions The 'Framework to achieve value in healthcare' provides a practical guide for hospitals to reduce non-value adding activities, improve the value of clinical care and reduce costs. Further research is indicated to establish its reliability in hospitals in other countries and hospitals that do not have an established PI program.

Digital technologies: An exploratory study of their role in the resilience of healthcare services.

Descriptions of resilient performance in healthcare services usually emphasize the role of skills and knowledge of caregivers. At the same time, the human factors discipline often frames digital technologies as sources of brittleness. This paper presents an exploratory investigation of the upside of ten digital technologies derived from Healthcare 4.0 (H4.0) in terms of their perceived contribution to six healthcare services and the four abilities of resilient healthcare: monitor, anticipate, respond, and learn. This contribution was assessed through a multinational survey conducted with 109 experts. Emergency rooms (ERs) and intensive care units (ICUs) stood out as the most benefited by H4.0 technologies. That is consistent with the high complexity of those services, which demand resilient performance. Four H4.0 technologies were top ranked regarding their impacts on the resilience of those services. They are further explored in follow-up interviews with ER and ICU professionals from hospitals in emerging and developed economies to collect examples of applications in their routines.

The aspartimide problem persists: Fluorenylmethyloxycarbonyl-solid-phase peptide synthesis (Fmoc-SPPS) chain termination due to formation of N-terminal piperazine-2,5-diones.

Aspartimide (Asi) formation is a notorious side reaction in peptide synthesis that is well characterized and described in literature. In this context, we observed significant amounts of chain termination in Fmoc-SPPS while synthesizing the N-terminal Xaa-Asp-Yaa motif. This termination was caused by the formation of piperazine-2,5-diones. We investigated this side reaction using a linear model peptide and independently synthesizing its piperazine-2,5-dione derivative. Nuclear magnetic resonance (NMR) data of the side product present in the crude linear peptide proves that exclusively the six-membered ring is formed whereas the theoretically conceivable seven-membered 1,4-diazepine-2,5-dione is not found. We propose a mechanism where nucleophilic attack of the N-terminal amino function takes place at the α-carbon of the carbonyl group of the corresponding Asi intermediate. In addition, we systematically investigated the impact of (a) different adjacent amino acid residues, (b) backbone protection, and (c) side chain protection of flanking amino acids. The side reaction is directly related to the Asi intermediate. Hence, hindering or avoiding Asi formation reduces or completely suppresses this side reaction.

Substitution determination of Fmoc-substituted resins at different wavelengths.

In solid-phase peptide synthesis, the nominal batch size is calculated using the starting resin substitution and the mass of the starting resin. The starting resin substitution constitutes the basis for the calculation of a whole set of important process parameters, such as the number of amino acid derivative equivalents. For Fmoc-substituted resins, substitution determination is often performed by suspending the Fmoc-protected starting resin in 20% (v/v) piperidine in DMF to generate the dibenzofulvene-piperidine adduct that is quantified by ultraviolet-visible spectroscopy. The spectrometric measurement is performed at the maximum absorption wavelength of the dibenzofulvene-piperidine adduct, that is, at 301.0 nm. The recorded absorption value, the resin weight and the volume are entered into an equation derived from Lambert-Beer's law, together with the substance-specific molar absorption coefficient at 301.0 nm, in order to calculate the nominal substitution. To our knowledge, molar absorption coefficients between 7100 l mol-1  cm-1 and 8100 l mol-1  cm-1 have been reported for the dibenzofulvene-piperidine adduct at 301.0 nm. Depending on the applied value, the nominal batch size may differ up to 14%. In this publication, a determination of the molar absorption coefficients at 301.0 and 289.8 nm is reported. Furthermore, proof is given that by measuring the absorption at 289.8 nm the impact of wavelength accuracy is reduced. © 2017 The Authors Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.

Quantitative assessment of preloaded 4-alkoxybenzyl alcohol resins for solid-phase peptide syntheses by 1D and 2D HR-MAS NMR.

The quality of preloaded Wang resins is very important for the success of solid-phase peptide syntheses (SPPS). A critical factor is the capping of remaining hydroxyl groups after loading with the first amino acid, since these free alcohols lead to truncated sequences during the following SPPS steps. Because the detection of hydroxyl groups by color tests is difficult and unreliable, the capping efficiency is often controlled by time-consuming peptide test syntheses. Here, we describe a two-dimensional, high resolution magic angle spinning NMR method for the quantitative determination of remaining 4-alkoxybenzyl alcohols in Fmoc-Xaa-Wang resins with a detection limit of 1 mol-%. The NMR method was validated with samples of known ratios between Fmoc-Ala-Wang and 4-alkoxybenzylalcohol resin. Application to a set of preloaded Fmoc-Ala- and Fmoc-Thr(tBu)-Wang test resins demonstrated that the full range of essential amino acids can be quantified without further spectrometer calibration. Compared to established test synthesis protocols, the NMR method represents not only advantages in terms of time and cost savings but also eliminates all inaccuracies due to further sample treatment like SPPS and cleavage from the resin.