Cysteine protecting groups: applications in peptide and protein science

Protecting group chemistry for the cysteine thiol group has enabled a vast array of peptide and protein chemistry over the last several decades. Increasingly sophisticated strategies for the protection, and subsequent deprotection, of cysteine have been developed, facilitating synthesis of complex disulfide-rich peptides, semisynthesis of proteins, and peptide/protein labelling in vitro and in vivo. In this review, we analyse and discuss the 60+ individual protecting groups reported for cysteine, highlighting their applications in peptide synthesis and protein science

Tyrosine bioconjugation - an emergent alternative

Protein bioconjugation is an increasingly important field of research, with wide-ranging applications in areas such as therapeutics and biomaterials. Traditional cysteine and lysine bioconjugation strategies are widely used and have been extensively researched, but in some cases they are not appropriate and alternatives are needed or they are not compatible with one another to enable the formation of dually (and distinctly) modified dual-conjugates (an increasingly desired class of bioconjugates). Here we review the heretofore less explored approach of tyrosine bioconjugation, which is rapidly becoming a constructive alternative/complement to the more well-established strategies. Herein we present an overview of the field, and then focus on promising recent methods that can achieve high conversion and chemoselectivity. This suggests that not only can tyrosine bioconjugation be used in conjunction with cysteine and lysine modification to obtain proteins with multiple different modifications, it is also becoming a stand-alone alternative to these more traditional methods

A Plug-and-Play Platform for the Formation of Trifunctional Cysteine Bioconjugates that also Offers Control over Thiol Cleavability

Linkers that enable the site-selective synthesis of chemically modified proteins are of great interest to the field of chemical biology. Homogenous bioconjugates often show advantageous pharmacokinetic profiles and consequently increased efficacy in vivo. Cysteine residues have been exploited as a route to site-selectively modify proteins, and many successfully approved therapeutics make use of cysteine directed conjugation reagents. However, commonly used linkers, including maleimide–thiol conjugates, are not stable to the low concentrations of thiol present in blood. Furthermore, only a few cysteine-targeting reagents enable the site-selective attachment of multiple functionalities: a useful tool in the fields of theranostics and therapeutic blood half-life extension. Herein, we demonstrate the application of the pyridazinedione motif to enable site-selective attachment of three functionalities to a protein bearing a single cysteine residue. Extending upon previously documented dual modification work, here we demonstrate that by exploiting a bromide leaving group as an additional reactive point on the pyridazinedione scaffold, a thiol or aniline derivative can be added to a protein, post-conjugation. Thiol cleavability appraisal of the resultant C–S and C–N linked thio-bioconjugates demonstrated C–S functionalized linkers to be cleavable and C–N functionalized linkers to be noncleavable when incubated in an excess of glutathione. The plug-and-play trifunctional platform was exemplified by attaching clinically relevant motifs: biotin, fluorescein, a polyethylene glycol chain, and a model peptide. This platform provides a rare opportunity to combine up to three functionalities on a protein in a site-selective fashion. Furthermore, by selecting the use of a thiol or an amine for functionalization, we provide unique control over linker cleavability toward thiols, allowing this novel linker to be applied in a range of physiological environments

Use of pyridazinediones as extracellular cleavable linkers through reversible cysteine conjugation

Herein we report a retro-Michael deconjugation pathway of thiol-pyridazinedione linked protein bioconjugates to provide a novel cleavable linker technology. We demonstrate that the novel pyridazinedione linker does not suffer from off-target modification with blood thiols (e.g., glutathione, human serum albumin (HSA)), which is in sharp contrast to an analogous maleimide linker

A novel thiol-labile cysteine protecting group for peptide synthesis based on a pyridazinedione (PD) scaffold

Herein we report a thiol-labile cysteine protecting group based on an unsaturated pyridazinedione (PD) scaffold. We establish compatibility of the PD in conventional solid phase peptide synthesis (SPPS), showcasing this in the on-resin synthesis of biologically relevant oxytocin. Furthermore, we establish the applicability of the PD protecting group towards both microwave-assisted SPPS and native chemical ligation (NCL) in a model system

Disulfide Modified IgG1: An Investigation of Biophysical Profile and Clinically Relevant Fc Interactions

Modification of immunoglobulin G (IgG) 1 proteins in cancer treatment is a rapidly growing field of research. Antibody-drug conjugates (ADCs) exploit the targeted nature of this immunotherapy by conjugating highly potent drugs to antibodies, allowing for effective transport of cargo(s) to cancerous cells. Of the many bioconjugation strategies now available for the formation of highly homogeneous ADCs, disulfide modification is considered an effective, low-cost, and widely accepted method for modifying IgG1s for improved clinical benefit. However, little is known about how disulfide modification impacts clinically relevant fragment crystallizable (Fc) region interactions. Although often overlooked as a secondary ADC function, Fc interactions could prove key in the rational design of cancer cell-targeting ADCs through consideration of potent mechanisms such as antibody-dependent cellular cytotoxicity (ADCC). This work explores different IgG1 disulfide modification techniques and the effect they have on quantifiable secondary IgG1 Fc interactions (e.g., CD16a and FcRn). The solvent accessible disulfide residues of trastuzumab, a clinically relevant IgG1, were modified to provide a range of bioconjugates with differing amounts of interchain covalent linkages. It was found that by natively rebridging the IgG1 model, all tested Fc functionalities were not significantly affected. Additionally, in non Fc-specific biophysical experiments (e.g., thermal stability/aggregation), the natively rebridged species provided an exceptional profile, showing no significant change from the tested native antibody. Conjugates with significant disruption of the covalent connectivity of IgG1 chains resulted in a suboptimal Fc profile (CD16a kinetics or ADCC activity), in addition to substandard non Fc-specific attributes (thermal stability). These results advocate native disulfide rebridging as an excellent synthetic strategy for forming homogeneous IgG1 bioconjugates, with no reported negative impact on biophysical profile relative to the native antibody

Microdevices for extensional rheometry of low viscosity elastic liquids : a review

Extensional flows and the underlying stability/instability mechanisms are of extreme relevance to the efficient operation of inkjet printing, coating processes and drug delivery systems, as well as for the generation of micro droplets. The development of an extensional rheometer to characterize the extensional properties of low viscosity fluids has therefore stimulated great interest of researchers, particularly in the last decade. Microfluidics has proven to be an extraordinary working platform and different configurations of potential extensional microrheometers have been proposed. In this review, we present an overview of several successful designs, together with a critical assessment of their capabilities and limitations

Factors affecting phage D29 infection: a tool to investigate different growth states of mycobacteria

Bacteriophages D29 and TM4 are able to infect a wide range of mycobacteria, including pathogenic and non pathogenic species. Successful phage infection of both fast- and slow-growing mycobacteria can be rapidly detected using the phage amplification assay. Using this method, the effect of oxygen limitation during culture of mycobacteria on the success of phage infection was studied. Both D29 and TM4 were able to infect cultures of M. smegmatis and Mycobacterium avium subspecies paratuberculosis (MAP) grown in liquid with aeration. However when cultures were grown under oxygen limiting conditions, only TM4 could productively infect the cells. Cell attachment assays showed that D29 could bind to the cells surface but did not complete the lytic cycle. The ability of D29 to productively infect the cells was rapidly recovered (within 1 day) when the cultures were returned to an aerobic environment and this recovery required de novo RNA synthesis. These results indicated that under oxygen limiting conditions the cells are entering a growth state which inhibits phage D29 replication, and this change in host cell biology which can be detected by using both phage D29 and TM4 in the phage amplification assay

Understanding the burden of interstitial lung disease post-COVID-19: the UK Interstitial Lung Disease-Long COVID Study (UKILD-Long COVID)

Introduction The COVID-19 pandemic has led to over 100 million cases worldwide. The UK has had over 4 million cases, 400 000 hospital admissions and 100 000 deaths. Many patients with COVID-19 suffer long-term symptoms, predominantly breathlessness and fatigue whether hospitalised or not. Early data suggest potentially severe long-term consequence of COVID-19 is development of long COVID-19-related interstitial lung disease (LC-ILD). Methods and analysis The UK Interstitial Lung Disease Consortium (UKILD) will undertake longitudinal observational studies of patients with suspected ILD following COVID-19. The primary objective is to determine ILD prevalence at 12 months following infection and whether clinically severe infection correlates with severity of ILD. Secondary objectives will determine the clinical, genetic, epigenetic and biochemical factors that determine the trajectory of recovery or progression of ILD. Data will be obtained through linkage to the Post-Hospitalisation COVID platform study and community studies. Additional substudies will conduct deep phenotyping. The Xenon MRI investigation of Alveolar dysfunction Substudy will conduct longitudinal xenon alveolar gas transfer and proton perfusion MRI. The POST COVID-19 interstitial lung DiseasE substudy will conduct clinically indicated bronchoalveolar lavage with matched whole blood sampling. Assessments include exploratory single cell RNA and lung microbiomics analysis, gene expression and epigenetic assessment. Ethics and dissemination All contributing studies have been granted appropriate ethical approvals. Results from this study will be disseminated through peer-reviewed journals. Conclusion This study will ensure the extent and consequences of LC-ILD are established and enable strategies to mitigate progression of LC-ILD

Alcohol, binge drinking and associated mental health problems in young urban Chileans

OBJECTIVE: To explore the link between alcohol use, binge drinking and mental health problems in a representative sample of adolescent and young adult Chileans. METHODS: Age and sex-adjusted Odds Ratios (OR) for four mental wellbeing measures were estimated with separate conditional logistic regression models for adolescents aged 15-20 years, and young adults aged 21-25 years, using population-based estimates of alcohol use prevalence rates from the Chilean National Health Survey 2010. RESULTS: Sixty five per cent of adolescents and 85% of young adults reported drinking alcohol in the last year and of those 83% per cent of adolescents and 86% of young adults reported binge drinking in the previous month. Adolescents who reported binging alcohol were also more likely, compared to young adults, to report being always or almost always depressed (OR 12.97 [95% CI, 1.86-19.54]) or to feel very anxious in the last month (OR 9.37 [1.77-19.54]). Adolescent females were more likely to report poor life satisfaction in the previous year than adolescent males (OR 8.50 [1.61-15.78]), feel always or almost always depressed (OR 3.41 [1.25-9.58]). Being female was also associated with a self-reported diagnosis of depression for both age groups (adolescents, OR 4.74 [1.49-15.08] and young adults, OR 4.08 [1.65-10.05]). CONCLUSION: Young people in Chile self-report a high prevalence of alcohol use, binge drinking and associated mental health problems. The harms associated with alcohol consumption need to be highlighted through evidence-based prevention programs. Health and education systems need to be strengthened to screen and support young people. Focussing on policy initiatives to limit beverage companies targeting alcohol to young people will also be needed