Chemo- and regio-selective differential modification of native cysteines on an antibody via the use of dehydroalanine forming reagents.

Protein modification has garnered increasing interest over the past few decades and has become an important tool in many aspects of chemical biology. In recent years, much effort has focused on site-selective modification strategies that generate more homogenous bioconjugates, and this is particularly so in the antibody modification space. Modifying native antibodies by targeting solvent-accessible cysteines liberated by interchain disulfide reduction is, perhaps, the predominant strategy for achieving more site-selectivity on an antibody scaffold. This is evidenced by numerous approved antibody therapeutics that have utilised cysteine-directed conjugation reagents and the plethora of methods/strategies focused on antibody cysteine modification. However, all of these methods have a common feature in that after the reduction of native solvent-accessible cystines, the liberated cysteines are all reacted in the same manner. Herein, we report the discovery and application of dehydroalanine forming reagents (including novel reagents) capable of regio- and chemo-selectively modifying these cysteines (differentially) on a clinically relevant antibody fragment and a full antibody. We discovered that these reagents could enable differential reactivity between light chain C-terminal cysteines, heavy chain hinge region cysteines (cysteines with an adjacent proline residue, Cys-Pro), and other heavy chain internal cysteines. This differential reactivity was also showcased on small molecules and on the peptide somatostatin. The application of these dehydroalanine forming reagents was exemplified in the preparation of a dually modified antibody fragment and full antibody. Additionally, we discovered that readily available amide coupling agents can be repurposed as dehydroalanine forming reagents, which could be of interest to the broader field of chemical biology.

Optimization of treatment workflow for 0.35T MR-Linac system.

PURPOSE: This study presents a novel and comprehensive framework for evaluating magnetic resonance guided radiotherapy (MRgRT) workflow by integrating the Failure Modes and Effects Analysis (FMEA) approach with Time-Driven Activity-Based Costing (TDABC). We assess the workflow for safety, quality, and economic implications, providing a holistic understanding of the MRgRT implementation. The aim is to offer valuable insights to healthcare practitioners and administrators, facilitating informed decision-making regarding the 0.35T MRIdian MR-Linac system's clinical workflow. METHODS: For FMEA, a multidisciplinary team followed the TG-100 methodology to assess the MRgRT workflow's potential failure modes. Following the mitigation of primary failure modes and workflow optimization, a treatment process was established for TDABC analysis. The TDABC was applied to both MRgRT and computed tomography guided RT (CTgRT) for typical five-fraction stereotactic body RT (SBRT) treatments, assessing total workflow and costs associated between the two treatment workflows. RESULTS: A total of 279 failure modes were identified, with 31 categorized as high-risk, 55 as medium-risk, and the rest as low-risk. The top 20% risk priority numbers (RPN) were determined for each radiation oncology care team member. Total MRgRT and CTgRT costs were assessed. Implementing technological advancements, such as real-time multi leaf collimator (MLC) tracking with volumetric modulated arc therapy (VMAT), auto-segmentation, and increasing the Linac dose rate, led to significant cost savings for MRgRT. CONCLUSION: In this study, we integrated FMEA with TDABC to comprehensively evaluate the workflow and the associated costs of MRgRT compared to conventional CTgRT for five-fraction SBRT treatments. FMEA analysis identified critical failure modes, offering insights to enhance patient safety. TDABC analysis revealed that while MRgRT provides unique advantages, it may involve higher costs. Our findings underscore the importance of exploring cost-effective strategies and key technological advancements to ensure the widespread adoption and financial sustainability of MRgRT in clinical practice.

A novel intranasal peptide vaccine inhibits non-small cell lung cancer with KRAS mutation.

KRAS mutations occur commonly in the lung and can lead to the development of non-small cell lung cancer (NSCLC). While the mutated KRAS protein is a neoantigen, it usually does not generate an effective anti-tumor immune response on mucosal/epithelial surfaces. Despite this, mutated KRAS remains a potential target for immunotherapy since immune targeting of this protein in animal models has been effective at eliminating tumor cells. We attempted to develop a KRAS vaccine using mutated and wild-type KRAS peptides in combination with a nanoemulsion (NE) adjuvant. The efficacy of this approach was tested in an inducible mutant KRAS-mouse lung tumor model. Animals were immunized intranasally using NE with KRAS peptides. These animals had decreased CD4+FoxP3+ T cells in both lymph nodes and spleen. Immunized animals also showed higher IFN-γ and IL-17a levels to mutated KRAS that were produced by CD8+ T cells and enhancement in KRAS-specific Th1 and Th17 responses that persisted for 3 months after the last vaccination. Importantly, the immunized animals had significantly decreased tumor incidence compared to control animals. In conclusion, a mucosal approach to KRAS vaccination demonstrated the ability to induce local KRAS-specific immune responses in the lung and resulted in reduced tumor incidence.

An Albumin-Holliday Junction Biomolecular Modular Design for Programmable Multifunctionality and Prolonged Circulation.

Combinatorial properties such as long-circulation and site- and cell-specific engagement need to be built into the design of advanced drug delivery systems to maximize drug payload efficacy. This work introduces a four-stranded oligonucleotide Holliday Junction (HJ) motif bearing functional moieties covalently conjugated to recombinant human albumin (rHA) to give a "plug-and-play" rHA-HJ multifunctional biomolecular assembly with extended circulation. Electrophoretic gel-shift assays show successful functionalization and purity of the individual high-performance liquid chromatography-purified modules as well as efficient assembly of the rHA-HJ construct. Inclusion of an epidermal growth factor receptor (EGFR)-targeting nanobody module facilitates specific binding to EGFR-expressing cells resulting in approximately 150-fold increased fluorescence intensity determined by flow cytometric analysis compared to assemblies absent of nanobody inclusion. A cellular recycling assay demonstrated retained albumin-neonatal Fc receptor (FcRn) binding affinity and accompanying FcRn-driven cellular recycling. This translated to a 4-fold circulatory half-life extension (2.2 and 0.55 h, for the rHA-HJ and HJ, respectively) in a double transgenic humanized FcRn/albumin mouse. This work introduces a novel biomolecular albumin-nucleic acid construct with extended circulatory half-life and programmable multifunctionality due to its modular design.

Creation of Polymer Datasets with Targeted Backbones for Screening of High-Performance Membranes for Gas Separation.

A simple approach was developed to computationally construct a polymer dataset by combining simplified molecular-input line-entry system (SMILES) strings of a targeted polymer backbone and a variety of molecular fragments. This method was used to create 14 polymer datasets by combining seven polymer backbones and molecules from two large molecular datasets (MOSES and QM9). Polymer backbones that were studied include four polydimethylsiloxane (PDMS) based backbones, poly(ethylene oxide) (PEO), poly(allyl glycidyl ether) (PAGE), and polyphosphazene (PPZ). The generated polymer datasets can be used for various cheminformatics tasks, including high-throughput screening for gas permeability and selectivity. This study utilized machine learning (ML) models to screen the polymers for CO2/CH4 and CO2/N2 gas separation using membranes. Several polymers of interest were identified. The results highlight that employing an ML model fitted to polymer selectivities leads to higher accuracy in predicting polymer selectivity compared to using the ratio of predicted permeabilities.

Results of a Highly Porous Metal-Backed Cementless Patella Implant: A Minimum 5-Year Follow-Up.

Initial design cementless metal-backed patellar implants failed due to multiple reasons including implant design, use of first-generation polyethylene, and surgical technique. This study evaluates clinical outcomes and survivorship of total knee arthroplasty (TKA) using a current generation highly porous metal-backed patellar component. One-hundred twenty-five consecutive primary cementless TKAs with a compression molded highly porous metal-backed patella were reviewed. One-hundred three TKAs (82.4%) with 5-year clinical and radiographic follow-up were available for review. These were matched with 103 consecutive TKAs using a cemented patella of the same implant design. The cementless cohort had a mean age of 65.5 years, body mass index (BMI) of 33.0, and follow-up of 64.4 months. Indications for cementless TKA were based on multiple factors including age, BMI, and bone quality. There were no revisions for loosening or mechanical failure of the cementless patella compared with two cemented patellae revised for aseptic loosening. Eight patients required revisions in the cementless cohort: three for prosthetic joint infection (PJI), two for instability, one periprosthetic femur fracture, one for patella instability, and one for extensor mechanism rupture. Five patients required revisions in the cemented cohort: two for aseptic patellar loosening, one for aseptic femoral loosening, one for PJI, and one for instability. All-cause survivorship at 5 years was 92.2 and 95.1% for the cementless metal-backed implant and cemented implant cohorts, respectively. Use of a compression molded highly porous metal-backed patella component demonstrated excellent clinical and radiographic results at 5-year follow-up. Longer follow-up is required to evaluate the ability of highly porous cementless patella implants to provide durable long-term fixation.

Comparison of clinical outcomes in patients undergoing one- and two-level minimally invasive lumbar microdiscectomy.

OBJECTIVE: Herniated nucleus pulposus (HNP) is one of the most common lumbar spine conditions treated surgically, often through a minimally invasive surgery (MIS) microdiscectomy approach. This technique attempts to reduce damage to the paraspinal muscular-ligamentous envelope. However, there are currently limited data regarding comparative outcomes using patient-reported outcome measures (PROMs) for one- and two-level MIS discectomies. The aim of this study was to quantify comparative clinical outcomes in patients undergoing one-level and two-level MIS lumbar microdiscectomy for HNP using PROMs. METHODS: The authors performed a retrospective review of patients undergoing MIS lumbar microdiscectomy between 2004 and 2019 for the primary diagnosis of HNP at a single academic institution. All patients had a minimum 1-year follow-up. Patient demographics and comorbidities were collected to establish baselines between cohorts. PROMs and minimal clinically important differences (MCIDs) were used to examine the patient's perception of operative success. Bivariate and multivariate linear/logistic regression analyses were used to compare one- and two-level discectomies. The bivariate analysis included the t-test and chi-square test, which were used to assess continuous and categorical variables, respectively. Statistical significance was established at p < 0.05. RESULTS: A total of 293 patients underwent one-level (n = 250) or two-level (n = 43) MIS discectomies. The mean follow-ups for the one- and two-level cohorts were 50.4 (SD 35.5) months and 61.6 (SD 39.8) months, respectively. Fewer female patients underwent two-level discectomies, and BMI and operative duration were higher in the two-level group (p < 0.001). Recurrent herniation requiring reoperation was recorded at rates of 6.80% and 11.6% in the one- and two-level groups, respectively (p = 0.270). Pre- and postoperative PROMs were largely similar between the cohorts; however, patients undergoing one-level discectomy had greater improvement in leg pain, and a significantly greater proportion of these patients achieved MCID for the leg pain visual analog scale score (p < 0.001). CONCLUSIONS: At the 1-year clinical follow-up, patients who underwent two-level discectomy had significantly less improvement in leg pain scores with lower achievement of MCID for leg pain improvement than patients undergoing one-level procedures. At the 1-year follow-up, there were no other significant differences in PROMs between the two cohorts. Given these findings, patients should be counseled regarding the anticipated outcomes to better manage expectations. Further studies are warranted to examine the long-term clinical outcomes associated with single- and multilevel MIS discectomy.

The Nitrile Bis-Thiol Bioconjugation Reaction.

Electron-poor aryl nitriles are promising reagents for bioconjugation due to their high electrophilicity and selectivity for reaction with thiols, albeit generally in a reversible manner. A transient species has previously been observed in such reactions, involving the addition of two thiols to the nitrile functional group, forming a tetrahedral amino dithioacetal (ADTA). In this work, the reaction of heteroaryl nitriles with bis-thiols is explored in an attempt to generate stable ADTAs, which could facilitate new bioconjugation protocols. By use of a 1,2-dithiol, or the incorporation of an electrophilic trap into the aryl nitrile design, the formation of stable products is achieved. The resultant "nitrile bis-thiol" (NBT) reaction is then explored in the context of protein modification, specifically to carry out antibody conjugation. By addition of these nitriles to the reduced disulfide bond of an antibody fragment, it is shown that, depending on the reagent design, cysteine-to-lysine transfer or disulfide bridged NBT products can be generated. Both represent site-selective conjugates and are shown to be stable when challenged with glutathione under physiological conditions and upon incubation in serum. Furthermore, the NBT reaction is tested in the more challenging context of a full antibody, and all four disulfide bonds are effectively modified by these new one-carbon bridging reagents. Overall, this reaction of heteroaryl-nitriles with bis-thiols is shown to be highly efficient and versatile, of tunable reversibility, and offers enticing prospects as a new addition to the toolbox of biocompatible "click"-type reactions.

Use of pyridazinediones for tuneable and reversible covalent cysteine modification applied to peptides, proteins and hydrogels.

Reversible cysteine modification has been found to be a useful tool for a plethora of applications such as selective enzymatic inhibition, activity-based protein profiling and/or cargo release from a protein or a material. However, only a limited number of reagents display reliable dynamic/reversible thiol modification and, in most cases, many of these reagents suffer from issues of stability, a lack of modularity and/or poor rate tunability. In this work, we demonstrate the potential of pyridazinediones as novel reversible and tuneable covalent cysteine modifiers. We show that the electrophilicity of pyridazinediones correlates to the rates of the Michael addition and retro-Michael deconjugation reactions, demonstrating that pyridazinediones provide an enticing platform for readily tuneable and reversible thiol addition/release. We explore the regioselectivity of the novel reaction and unveil the reason for the fundamental increased reactivity of aryl bearing pyridazinediones by using DFT calculations and corroborating findings with SCXRD. We also applied this fundamental discovery to making more rapid disulfide rebridging agents in related work. We finally provide the groundwork for potential applications in various areas with exemplification using readily functionalised "clickable" pyridazinediones on clinically relevant cysteine and disulfide conjugated proteins, as well as on a hydrogel material.

Cementless metal-backed patellar components in primary total knee arthroplasty using an implant of modern design.

Aims: The use of cementless total knee arthroplasty (TKA) components has increased during the past decade. The initial design of cementless metal-backed patellar components had shown high failure rates due to many factors. The aim of this study was to evaluate the clinical results of a second-generation cementless, metal-backed patellar component of a modern design. Methods: This was a retrospective review of 707 primary TKAs in 590 patients from a single institution, using a cementless, metal-backed patellar component with a mean follow-up of 6.9 years (2 to 12). A total of 409 TKAs were performed in 338 females and 298 TKAs in 252 males. The mean age of the patients was 63 years (34 to 87) and their mean BMI was 34.3 kg/m2 (18.8 to 64.5). The patients were chosen to undergo a cementless procedure based on age and preoperative radiological and intraoperative bone quality. Outcome was assessed using the Knee Society knee and function scores and range of motion (ROM), complications, and revisions. Results: A total of 24 TKAs (3.4%) in 24 patients failed and required revision surgery, of which five were due to patellar complications (0.71%): one for aseptic patellar loosening (0.14%) and four for polyethylene dissociation (0.57%). A total of 19 revisions (2.7%) were undertaken in 19 patients for indications which did not relate to the patella: four for aseptic tibial loosening (0.57%), one for aseptic femoral loosening (0.14%), nine for periprosthetic infection (1.3%), one for popliteus impingement (0.14%), and four for instability (0.57%). Knee Society knee and function scores, and ROM, improved significantly when comparing pre- and postoperative values. Survival of the metal-backed patellar component for all-cause failure was 97.5% (95% confidence interval 94.9% to 100%) at 12 years. Conclusion: The second-generation cementless TKA design of metal-backed patellar components showed a 97.5% survival at 12 years, with polyethylene dissociation from the metal-backing being the most common cause of patellar failure. In view of the increased use of TKA, especially in younger, more active, or obese patients, these findings are encouraging at mean follow-up of seven years.

Delicate Hybrid Laponite-Cyclic Poly(ethylene glycol) Nanoparticles as a Potential Drug Delivery System.

The objective of the study was to explore the feasibility of a new drug delivery system using laponite (LAP) and cyclic poly(ethylene glycol) (cPEG). Variously shaped and flexible hybrid nanocrystals were made by both the covalent and physical attachment of chemically homogeneous cyclized PEG to laponite nanodisc plates. The size of the resulting, nearly spherical particles ranged from 1 to 1.5 µm, while PEGylation with linear methoxy poly (ethylene glycol) (mPEG) resulted in fragile sheets of different shapes and sizes. When infused with 10% doxorubicin (DOX), a drug commonly used in the treatment of various cancers, the LAP-cPEG/DOX formulation was transparent and maintained liquid-like homogeneity without delamination, and the drug loading efficiency of the LAP-cPEG nano system was found to be higher than that of the laponite-poly(ethylene glycol) LAP-mPEG system. Furthermore, the LAP-cPEG/DOX formulation showed relative stability in phosphate-buffered saline (PBS) with only 15% of the drug released. However, in the presence of human plasma, about 90% of the drug was released continuously over a period of 24 h for the LAP-cPEG/DOX, while the LAP-mPEG/DOX formulation released 90% of DOX in a 6 h burst. The results of the cell viability assay indicated that the LAP-cPEG/DOX formulation could effectively inhibit the proliferation of A549 lung carcinoma epithelial cells. With the DOX concentration in the range of 1-2 µM in the LAP-cPEG/DOX formulation, enhanced drug effects in both A549 lung carcinoma epithelial cells and primary lung epithelial cells were observed compared to LAP-mPEG/DOX. The unique properties and effects of cPEG nanoparticles provide a potentially better drug delivery system and generate interest for further targeting studies and applications.

Availability of palliative parenteral nutrition to patients with advanced cancer: A national survey of service provision.

BACKGROUND: Patients with advanced malignancy who are unable to meet their nutritional requirements orally or enterally as a result of intestinal failure may be considered for parenteral nutrition support. Current UK guidance recommends that patients with a 3-month prognosis and good performance status (i.e., Karnofsky performance status >50) should be considered for this intervention at home (termed Home Parenteral Nutrition; HPN). However, HPN is a nationally commissioned service by National Health Service (NHS) England and Improvement that can only be initiated at specific NHS centres and so may not be easily accessed by patients outside of these centres. This survey aimed to identify current clinical practice across UK hospitals about how palliative parenteral nutrition is initiated. METHODS: Clinical staff associated with Nutrition Support Teams at NHS Organisations within the UK were invited to complete an electronically administered survey of national clinical practice through advertisements posted on relevant professional interest groups. RESULTS: Sixty clinicians responded to the survey administered between September and November 2020. The majority of respondents responded positively that decisions made to initiate palliative parenteral nutrition were conducted in alignment with current national guidance in relation to decision-making and formulation of parenteral nutrition. Variation was observed in relation to the provision of advance care planning in relation to nutrition support prior to discharge, as well as the consideration of venting gastrostomy placement in patients with malignant bowel obstruction unsuitable for surgical intervention. CONCLUSIONS: Adherence to current national guidance in relation to the provision of palliative parenteral nutrition is variable for some aspects of care. Further work is required particularly in relation to maximising the opportunity for the provision of advance care planning prior to discharge in this patient cohort.

GC-MS Techniques Investigating Potential Biomarkers of Dying in the Last Weeks with Lung Cancer.

Predicting when a patient with advanced cancer is dying is a challenge and currently no prognostic test is available. We hypothesised that a dying process from cancer is associated with metabolic changes and specifically with changes in volatile organic compounds (VOCs). We analysed urine from patients with lung cancer in the last weeks of life by headspace gas chromatography mass spectrometry. Urine was acidified or alkalinised before analysis. VOC changes in the last weeks of life were identified using univariate, multivariate and linear regression analysis; 12 VOCs increased (11 from the acid dataset, 2 from the alkali dataset) and 25 VOCs decreased (23 from the acid dataset and 3 from the alkali dataset). A Cox Lasso prediction model using 8 VOCs predicted dying with an AUC of 0.77, 0.78 and 0.85 at 30, 20 and 10 days and stratified patients into a low (median 10 days), medium (median 50 days) or high risk of survival. Our data supports the hypothesis there are specific metabolic changes associated with the dying. The VOCs identified are potential biomarkers of dying in lung cancer and could be used as a tool to provide additional prognostic information to inform expert clinician judgement and subsequent decision making.

Endplate abnormalities, Modic changes and their relationship to alignment parameters and surgical outcomes in the cervical spine.

Modic changes (MC) and endplate abnormalities (EA) have been shown to impact preoperative symptoms and outcomes following spinal surgery. However, little is known about how these phenotypes impact cervical alignment. This study aimed to evaluate the impact that these phenotypes have on preoperative, postoperative, and changes in cervical alignment in patients undergoing anterior cervical discectomy and fusion (ACDF). We performed a retrospective study of prospectively collected data of ACDF patients at a single institution. Preoperative magnetic resonance imagings (MRIs) were used to assess for the MC and EA. Patients were subdivided into four groups: MC-only, EA-only, the combined Modic-Endplate-Complex (MEC), and patients without either phenotype. Pre and postoperative MRIs were used to assess alignment parameters. Associations with imaging phenotypes and alignment parameters were assessed, and statistical significance was set at p < 0.5. A total of 512 patients were included, with 84 MC-only patients, 166 EA-only patients, and 71 patients with MEC. Preoperative MC (p = 0.031) and the MEC (p = 0.039) had significantly lower preoperative T1 slope compared to controls. Lower preoperative T1 slope was a risk factor for MC (p = 0.020) and MEC (p = 0.029) and presence of MC (Type II) and the MEC (Type III) was predictive of lower preoperative T1 slope. There were no differences in postoperative alignment measures or patient reported outcome measures. MC and endplate pathologies such as the MEC appear to be associated with worse cervical alignment at baseline relative to patients without these phenotypes. Poor alignment may be an adaptive response to these degenerative findings or may be a risk factor for their development.

Dual reactivity disulfide bridging reagents; enabling new approaches to antibody fragment bioconjugation.

Disulfide bridging, also known as disulfide stapling, is a powerful strategy for the construction of site-selective protein bioconjugates. Here we describe the first examples of a new class of such reagents, containing a 'stable-labile' design. These dual-reactive reagents are designed to form a stable bond to one cysteine and a labile bond to the second; resulting in a robust attachment to the protein with one end of the bridge, whilst the other end serves as a reactive handle for subsequent bioconjugation. By incorporating thioesters into these bridges, we demonstrate that they are primed for native chemical ligation (NCL) with N-terminal cysteines; offering an alternative to the requirement for C-terminal thioesters for use in such ligations. Alternatively, the use of hydrazine as the ligating nucleophile enables a separate cargo to be attached to each cysteine residue, which are exploited to insert variably cleavable linkers. These methodologies are demonstrated on an antibody fragment, and serve to expand the scope of disulfide bridging strategies whilst offering a convenient route to the construction of multifunctional antibody fragment conjugates.

Correction: Use of pyridazinediones as extracellular cleavable linkers through reversible cysteine conjugation.

Correction for 'Use of pyridazinediones as extracellular cleavable linkers through reversible cysteine conjugation' by Calise Bahou et al., Chem. Commun., 2019, 55, 14829-14832, https://doi.org/10.1039/C9CC08362F.

Differential responses of COPD macrophages to respiratory bacterial pathogens.

COPD patients have increased susceptibility to airway bacterial colonisation. Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae are three of the most common respiratory bacterial species in COPD. H. influenzae colonisation, but not other bacteria, in COPD patients is associated with higher sputum neutrophil counts. Alveolar macrophages are key in clearance of bacteria as well as releasing mediators to recruit and activate other immune cells in response to infection. The aim was to characterise differences in COPD macrophage responses to H. influenzae, M. catarrhalis and S. pneumoniae, focusing on release of inflammatory and chemotactic mediators, and apoptosis regulation. Lung macrophages and monocyte-derived macrophages from COPD patients and control subjects were exposed to H. influenzae, M. catarrhalis or S. pneumoniae. Cytokine secretion (tumour necrosis factor-α, interleukin (IL)-6, CXCL8, CCL5 and IL-1ß) were measured by ELISA and quantitative reverse transcriptase PCR (RT-qPCR), and apoptosis genes MCL-1, BCL-2, BAX and BAK1 by RT-qPCR. Apoptosis and reactive oxygen species (ROS) release were also measured. Macrophages responded differentially to the bacterial species, with increased, prolonged production of the neutrophil chemoattractant CXCL8 in response to H. influenzae and M. catarrhalis but not S. pneumoniae. S. pneumoniae initiated macrophage apoptosis and ROS release, H. influenzae and M. catarrhalis did not and increased anti-apoptosis gene expression (BCL-2 5.5-fold and MCL-1 2.4-fold, respectively). Differential cytokine responses of macrophages to these bacterial species can explain neutrophilic airway inflammation associated with H. influenzae, but not S. pneumoniae in COPD. Furthermore, delayed macrophage apoptosis is a potential mechanism contributing to inability to clear H. influenzae.

Unearthing the unique stability of thiophosphonium-C-terminal cysteine adducts on peptides and proteins.

Herein we report a fundamental discovery on the use of tris(dialkylamino)phosphine reagents for peptide and protein modification. We discovered that C-terminal thiophosphonium species, which are uniquely stable, could be selectively and rapidly generated from their disulfide counterparts. In sharp and direct contrast, internal thiophosphonium species rapidly degrade to dehydroalanine. We demonstrate this remarkable chemoselectivity on a bis-cysteine model peptide, and the formation of a stable C-terminal-thiophosphonium adduct on an antibody fragment, as well as characterise the species in various small molecule/peptide studies.

Albumin Biomolecular Drug Designs Stabilized through Improved Thiol Conjugation and a Modular Locked Nucleic Acid Functionalized Assembly.

Albumin-nucleic acid biomolecular drug designs offer modular multifunctionalization and extended circulatory half-life. However, stability issues associated with conventional DNA nucleotides and maleimide bioconjugation chemistries limit the clinical potential. This work aims to improve the stability of this thiol conjugation and nucleic acid assembly by employing a fast-hydrolyzing monobromomaleimide (MBM) linker and nuclease-resistant nucleotide analogues, respectively. The biomolecular constructs were formed by site-selective conjugation of a 12-mer oligonucleotide to cysteine 34 (Cys34) of recombinant human albumin (rHA), followed by annealing of functionalized complementary strands bearing either a fluorophore or the cytotoxic drug monomethyl auristatin E (MMAE). Formation of conjugates and assemblies was confirmed by gel shift analysis and mass spectrometry, followed by investigation of serum stability, neonatal Fc receptor (FcRn)-mediated cellular recycling, and cancer cell killing. The MBM linker afforded rapid conjugation to rHA and remained stable during hydrolysis. The albumin-nucleic acid biomolecular assembly composed of stabilized oligonucleotides exhibited high serum stability and retained FcRn engagement mediating FcRn-mediated cellular recycling. The MMAE-containing assembly exhibited cytotoxicity in the human MIA PaCa-2 pancreatic cancer cell line with an IC50 of 342 nM, triggered by drug release from breakdown of an acid-labile linker. In summary, this work presents rHA-nucleic acid module-based assemblies with improved stability and retained module functionality that further promotes the drug delivery potential of this biomolecular platform.

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.