Cross aldol OPAL bioconjugation outcompetes intramolecular hemiaminal cyclisation of proline adjacent N-terminal α-oxo aldehydes at acidic pH.

Novel methods to construct small molecule-protein bioconjugates are integral to the development of new biomedicines for a variety of diseases. C-C linked bioconjugates are increasingly desirable in this application due to their in vivo stability and can be accessed through cross aldol bioconjugation of reactive α-oxo aldehyde handles easily introduced at the N-terminus of proteins by periodate oxidation. We previously developed an organocatalyst-mediated protein aldol ligation (OPAL) for chemical modification of these reactive aldehydes, but the efficiency of this method was limited when a proline residue was directly adjacent to the N-terminus due to intramolecular hemiaminal formation. Herein we explore the competition between this cyclisation and the OPAL modification and demonstrate bioconjugation can be favoured through use of acidic pH for both oxidation and OPAL, and optimisation of reaction conditions and organocatalyst. We then showcase the utility of this acidic-OPAL in modification of the cholera toxin B-subunit (CTB), a homo-pentameric protein of biomedical promise.

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.

Hydrogel Cross-Linking via Thiol-Reactive Pyridazinediones.

Thiol-reactive Michael acceptors are commonly used for the formation of chemically cross-linked hydrogels. In this paper, we address the drawbacks of many Michael acceptors by introducing pyridazinediones as new cross-linking agents. Through the use of pyridazinediones and their mono- or dibrominated analogues, we show that the mechanical strength, swelling ratio, and rate of gelation can all be controlled in a pH-sensitive manner. Moreover, we demonstrate that the degradation of pyridazinedione-gels can be induced by the addition of thiols, thus providing a route to responsive or dynamic gels, and that monobromo-pyridazinedione gels are able to support the proliferation of human cells. We anticipate that our results will provide a valuable and complementary addition to the existing toolkit of cross-linking agents, allowing researchers to tune and rationally design the properties of biomedical hydrogels.

Recent advances in N- and C-terminus cysteine protein bioconjugation.

Advances in the site-specific chemical modification of proteins, also referred to as protein bioconjugation, have proved instrumental in revolutionary approaches to designing new protein-based therapeutics. Of the sites available for protein modification, cysteine residues or the termini of proteins have proved especially popular owing to their favorable properties for site-specific modification. Strategies that, therefore, specifically target cysteine at the termini offer a combination of these favorable properties of cysteine and termini bioconjugation. In this review, we discuss these strategies with a particular focus on those reported recently and provide our opinion on the future direction of the field.

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.

Correction: Tyrosine bioconjugation - an emergent alternative.

Correction for 'Tyrosine bioconjugation - an emergent alternative' by Peter A. Szijj et al., Org. Biomol. Chem., 2020, 18, 9018-9028, https://doi.org/10.1039/D0OB01912G.

Functionalisation of ethereal-based saturated heterocycles with concomitant aerobic C-H activation and C-C bond formation.

With an ever-growing emphasis on sustainable synthesis, aerobic C-H activation (the use of oxygen in air to activate C-H bonds) represents a highly attractive conduit for the development of novel synthetic methodologies. Herein, we report the air mediated functionalisation of various saturated heterocycles and ethers via aerobically generated radical intermediates to form new C-C bonds using acetylenic and vinyl triflones as radical acceptors. This enables access to a variety of acetylenic and vinyl substituted saturated heterocycles that are rich in synthetic value. Mechanistic studies and control reactions support an aerobic radical-based C-H activation mechanism.

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.

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.

A Tale of Two Bioconjugations: pH Controlled Divergent Reactivity of Protein α-oxo-Aldehydes in Competing α-oxo-Mannich and Catalyst-Free Aldol Ligations.

Site-selective chemical methods for protein bioconjugation have revolutionized the fields of cell and chemical biology through the development of novel protein/enzyme probes bearing fluorescent, spectroscopic, or even toxic cargos. Herein, we report two new methods for the bioconjugation of α-oxo aldehyde handles within proteins using small molecule aniline and/or phenol probes. The "α-oxo-Mannich" and "catalyst-free aldol" ligations both compete for the electrophilic α-oxo aldehyde, which displays pH divergent reactivity proceeding through the "Mannich" pathway at acidic pH to afford bifunctionalized bioconjugates, and the "catalyst-free aldol" pathway at neutral pH to afford monofunctionalized bioconjugates. We explore the substrate scope and utility of both of these bioconjugations in the construction of neoglycoproteins, in the process formulating a mechanistic rationale for how both pathways intersect with each other at different reaction pH's.

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.

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.

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.

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.

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.

Site-selective C-C modification of proteins at neutral pH using organocatalyst-mediated cross aldol ligations.

The bioconjugation of proteins with small molecules has proved an invaluable strategy for probing and perturbing biological mechanisms. The general use of chemical methods for protein functionalisation can be limited however by the requirement for complicated reaction partners to be present in large excess, and harsh conditions which are incompatible with many protein scaffolds. Herein we describe a site-selective organocatalyst-mediated protein aldol ligation (OPAL) that affords stable carbon-carbon linked bioconjugates at neutral pH. OPAL enables rapid modification of proteins using simple aldehyde probes in minimal excess, and is utilised here in the affinity tagging of proteins in cell lysate. Furthermore we demonstrate that the ß-hydroxy aldehyde OPAL product can be functionalised again at neutral pH in a tandem organocatalyst-mediated oxime ligation. This tandem strategy is showcased in the 'chemical mimicry' of a previously inaccessible natural dual post-translationally modified protein integral to the pathogenesis of the neglected tropical disease Leishmaniasis.

Bacteroides thetaiotaomicron generates diverse α-mannosidase activities through subtle evolution of a distal substrate-binding motif.

A dominant human gut microbe, the well studied symbiont Bacteroides thetaiotaomicron (Bt), is a glyco-specialist that harbors a large repertoire of genes devoted to carbohydrate processing. Despite strong similarities among them, many of the encoded enzymes have evolved distinct substrate specificities, and through the clustering of cognate genes within operons termed polysaccharide-utilization loci (PULs) enable the fulfilment of complex biological roles. Structural analyses of two glycoside hydrolase family 92 α-mannosidases, BT3130 and BT3965, together with mechanistically relevant complexes at 1.8-2.5 Šresolution reveal conservation of the global enzyme fold and core catalytic apparatus despite different linkage specificities. Structure comparison shows that Bt differentiates the activity of these enzymes through evolution of a highly variable substrate-binding region immediately adjacent to the active site. These observations unveil a genetic/biochemical mechanism through which polysaccharide-processing bacteria can evolve new and specific biochemical activities from otherwise highly similar gene products.

Palladium-unleashed proteins: gentle aldehyde decaging for site-selective protein modification.

Protein bioconjugation frequently makes use of aldehydes as reactive handles, with methods for their installation being highly valued. Here a new, powerful strategy to unmask a reactive protein aldehyde is presented. A genetically encoded caged glyoxyl aldehyde, situated in solvent-accessible locations, can be rapidly decaged through treatment with just one equivalent of allylpalladium(ii) chloride dimer at physiological pH. The protein aldehyde can undergo subsequent oxime ligation for site-selective protein modification. Quick yet mild conditions, orthogonality and powerful exposed reactivity make this strategy of great potential in protein modification.

Site-selective incorporation and ligation of protein aldehydes.

The incorporation of aldehyde handles into proteins, and subsequent chemical reactions thereof, is rapidly proving to be an effective way of generating homogeneous, covalently linked protein constructs that can display a vast array of functionality. In this review, we discuss methods for introducing aldehydes into target proteins, and summarise the ligation strategies for site-selective modification of proteins containing this class of functional handles.