Increasing the versatility of the biphenyl-fused-dioxacyclodecyne class of strained alkynes.

Biphenyl-fused-dioxacyclodecynes are a promising class of strained alkyne for use in Cu-free 'click' reactions. In this paper, a series of functionalised derivatives of this class of reagent, containing fluorescent groups, are described. Studies aimed at understanding and increasing the reactivity of the alkynes are also presented, together with an investigation of the bioconjugation of the reagents with an azide-labelled protein.

All-in-one disulfide bridging enables the generation of antibody conjugates with modular cargo loading.

Antibody-drug conjugates (ADCs) are valuable therapeutic entities which leverage the specificity of antibodies to selectively deliver cytotoxins to antigen-expressing targets such as cancer cells. However, current methods for their construction still suffer from a number of shortcomings. For instance, using a single modification technology to modulate the drug-to-antibody ratio (DAR) in integer increments while maintaining homogeneity and stability remains exceptionally challenging. Herein, we report a novel method for the generation of antibody conjugates with modular cargo loading from native antibodies. Our approach relies on a new class of disulfide rebridging linkers, which can react with eight cysteine residues, thereby effecting all-in-one bridging of all four interchain disulfides in an IgG1 antibody with a single linker molecule. Modification of the antibody with the linker in a 1 : 1 ratio enabled the modulation of cargo loading in a quick and selective manner through derivatization of the linker with varying numbers of payload attachment handles to allow for attachment of either 1, 2, 3 or 4 payloads (fluorescent dyes or cytotoxins). Assessment of the biological activity of these conjugates demonstrated their exceptional stability in human plasma and utility for cell-selective cytotoxin delivery or imaging/diagnostic applications.

Rapid and robust cysteine bioconjugation with vinylheteroarenes.

Methods for residue-selective and stable modification of canonical amino acids enable the installation of distinct functionality which can aid in the interrogation of biological processes or the generation of new therapeutic modalities. Herein, we report an extensive investigation of reactivity and stability profiles for a series of vinylheteroarene motifs. Studies on small molecule and protein substrates identified an optimum vinylheteroarene scaffold for selective cysteine modification. Utilisation of this lead linker to modify a number of protein substrates with various functionalities, including the synthesis of a homogeneous, stable and biologically active antibody-drug conjugate (ADC) was then achieved. The reagent was also efficient in labelling proteome-wide cysteines in cell lysates. The efficiency and selectivity of these reagents as well as the stability of the products makes them suitable for the generation of biotherapeutics or studies in chemical biology.

Site-selective modification strategies in antibody-drug conjugates.

Antibody-drug conjugates (ADCs) harness the highly specific targeting capabilities of an antibody to deliver a cytotoxic payload to specific cell types. They have garnered widespread interest in drug discovery, particularly in oncology, as discrimination between healthy and malignant tissues or cells can be achieved. Nine ADCs have received approval from the US Food and Drug Administration and more than 80 others are currently undergoing clinical investigations for a range of solid tumours and haematological malignancies. Extensive research over the past decade has highlighted the critical nature of the linkage strategy adopted to attach the payload to the antibody. Whilst early generation ADCs were primarily synthesised as heterogeneous mixtures, these were found to have sub-optimal pharmacokinetics, stability, tolerability and/or efficacy. Efforts have now shifted towards generating homogeneous constructs with precise drug loading and predetermined, controlled sites of attachment. Homogeneous ADCs have repeatedly demonstrated superior overall pharmacological profiles compared to their heterogeneous counterparts. A wide range of methods have been developed in the pursuit of homogeneity, comprising chemical or enzymatic methods or a combination thereof to afford precise modification of specific amino acid or sugar residues. In this review, we discuss advances in chemical and enzymatic methods for site-specific antibody modification that result in the generation of homogeneous ADCs.

Synthesis and Characterization of Dendrimer-Based Polysarcosine Star Polymers: Well-Defined, Versatile Platforms Designed for Drug-Delivery Applications.

This paper describes the synthesis of star polymers designed for future drug-delivery applications. A generation-5 lysine dendrimer was used as a macroinitiator for the ring-opening polymerization of the sarcosine N-carboxyanhydride monomer to produce 32-arm star polymers with narrow molar mass distributions and desirable hydrodynamic size control. Fluorescent dye-labeled polymers were dosed in mice to measure plasma pharmacokinetics. Long circulation times were observed, representing ideal properties for biophysical targeting of tumors. In vivo efficacy of one of these star polymers conjugated to the therapeutic molecule SN-38 was evaluated in mice bearing SW620 xenografted tumors to demonstrate high antitumor activity and low body weight loss compared to the SN-38 prodrug irinotecan and this shows the potential of these delivery systems. As a further build, we demonstrated that these star polymers can be easily chain-end-functionalized with useful chemical moieties, giving opportunities for future receptor-targeting strategies. Finally, we describe the synthetic advantages of these star polymers that make them attractive from a pharmaceutical manufacturing perspective and report characterization of the polymers with a variety of techniques.

General dual functionalisation of biomacromolecules via a cysteine bridging strategy.

Site-selective modification of peptides and proteins has resulted in the development of a host of novel tools for the study of cellular systems or the synthesis of enhanced biotherapeutics. There is a need for useful methodologies that enable site-selective modification of native peptides or proteins, which is even more prevalent when modification of the biomolecule with multiple payloads is desired. Herein, we report the development of a novel dual functional divinylpyrimidine (dfDVP) platform that enables robust and modular modification of peptides, antibody fragments and antibodies. These biomacromolecules could be easily functionalised with a range of functional payloads (e.g. fluorescent dyes, cytotoxic warheads or cell-penetrating tags). Importantly, the dual functionalised peptides and antibodies demonstrated exquisite bioactivity in a range of in vitro cellular assays, showcasing the enhanced utility of these bioactive conjugates.

Total synthesis and biological evaluation of simplified aplyronine analogues as synthetically tractable anticancer agents.

The aplyronines are a family of highly cytotoxic marine natural products with potential application in targeted cancer chemotherapy. To address the severe supply issue, function-oriented molecular editing of their macrolactone scaffold led to the design of a series of simplified aplyronine analogues. Enabled by a highly convergent aldol-based route, the total synthesis of four analogues was achieved, with a significant improvement in step economy versus previous compounds, and their cancer cell growth inhibition in the HeLa cell line was determined. The modular strategy presented offers a means for significantly shortening their chemical synthesis to facilitate the continued development of this promising class of anticancer agent.

Cleavable linkers in antibody-drug conjugates.

Antibody-Drug Conjugates (ADCs) are now established as a major class of therapeutics for the clinical treatment of cancer. The properties of the linker between the antibody and the payload are proven to be critical to the success of an ADC. Although ADC linkers can be 'non-cleavable', the vast majority of ADCs in clinical development have specific release mechanisms to allow controlled linker cleavage at the target site and are thus termed 'cleavable'. In recent years, the development of new methods of drug release from ADCs has continued in parallel to the deepening understanding of the biological processes underlying the mechanisms of action of pre-existing technologies. This review summarises the advances in the field of cleavable linker technologies for ADCs.

Macrocyclisation and functionalisation of unprotected peptides via divinyltriazine cysteine stapling.

We report a novel divinyltriazine linker for the stapling of two cysteine residues to form macrocyclic peptides from their unprotected linear counterparts. The stapling reaction occurred rapidly under mild conditions on a range of unprotected peptide sequences. The resulting constrained peptides displayed greater stability in a serum stability assay when compared to their linear counterparts.

A general approach for the site-selective modification of native proteins, enabling the generation of stable and functional antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are a class of targeted therapeutics that utilize the specificity of antibodies to selectively deliver highly potent cytotoxins to target cells. Although recent years have witnessed significant interest in ADCs, problems remain with the standard linkage chemistries used for cytotoxin-antibody bioconjugation. These typically (1) generate unstable constructs, which may lead to premature cytotoxin release, (2) often give a wide variance in drug-antibody ratios (DAR) and (3) have poor control of attachment location on the antibody, resulting in a variable pharmacokinetic profile. Herein, we report a novel divinylpyrimidine (DVP) linker platform for selective bioconjugation via covalent re-bridging of reduced disulfide bonds on native antibodies. Model studies using the non-engineered trastuzumab antibody validate the utility of this linker platform for the generic generation of highly plasma-stable and functional antibody constructs that incorporate variable biologically relevant payloads (including cytotoxins) in an efficient and site-selective manner with precise control over DAR. DVP linkers were also used to efficiently re-bridge both monomeric and dimeric protein systems, demonstrating their potential utility for general protein modification, protein stabilisation or the development of other protein-conjugate therapeutics.

Correction: A general approach for the site-selective modification of native proteins, enabling the generation of stable and functional antibody-drug conjugates.

[This corrects the article DOI: 10.1039/C8SC04645J.].

An Alternative Focus for Route Design for the Synthesis of Antibody-Drug Conjugate Payloads.

An analysis of Antibody-Drug Conjugate Payload manufacturing has revealed that the majority of the cost is associated with the use of high-containment facilities for the latter stages of the synthesis. To make a significant reduction in the Cost of Goods (CoGs), a new approach to route design has been introduced which focuses on minimizing the number of steps that require high containment. This approach has been exemplified in a new synthesis of tesirine, including the first application of a ring-closing copper(I)/TEMPO aerobic oxidation to the pyrrolobenzodiazepine ring system, affording a 60% reduction in CoGs.

A strained alkyne-containing bipyridine reagent; synthesis, reactivity and fluorescence properties.

We report the synthesis of a bipyridyl reagent containing a strained alkyne, which significantly restricts its flexibility. Upon strain-promoted alkyne-azide cycloaddition (SPAAC) with an azide, which does not require a Cu catalyst, the structure becomes significantly more flexible and an increase in fluorescence is observed. Upon addition of Zn(ii), the fluorescence is enhanced further. The reagent has the potential to act as a fluorescent labelling agent with azide-containing substrates, including biological molecules.

Synthesis and cycloaddition reactions of strained alkynes derived from 2,2'-dihydroxy-1,1'-biaryls.

A series of strained alkynes, based on the 2,2'-dihydroxy-1,1'-biaryl structure, were prepared in a short sequence from readily-available starting materials. These compounds can be readily converted into further derivatives including examples containing fluorescent groups with potential for use as labelling reagents. The alkynes are able to react in cycloadditions with a range of azides without the requirement for a copper catalyst, in clean reactions with no observable side reactions.

Concise copper-catalyzed synthesis of tricyclic biaryl ether-linked aza-heterocyclic ring systems.

A new method for the synthesis of tricyclic biaryl ether-linked ring systems incorporating seven-, eight-, and nine-membered ring amines is presented. In the presence of catalytic quantities of copper(I), readily accessible acyclic precursors undergo an intramolecular carbon-oxygen bond-forming reaction facilitated by a "templating" chelating nitrogen atom. The methodology displays a broad substrate scope, is practical, and generates rare and biologically interesting tricyclic heteroaromatic products that are difficult to access by other means.

Spiro-fused pyrrolidine, piperidine, and oxindole scaffolds from lactams.

Expedient routes to three classes of novel spiro-fused pyrrolidine, piperidine, and indoline heterocycle scaffolds are described. These three-dimensional frameworks, which conform to the "rule of three", are suitably protected to allow for site-selective manipulation and functionalization. Different modes of substrate control were explored, which allow for good to excellent levels of diastereoselectivity and dispense with the need for additional chiral reagents or catalysts. The concepts developed were applied in short, formal syntheses of (±)-coerulescine and (±)-horsfiline.

Manganese(III) acetate mediated oxidative radical cyclizations. Toward vicinal all-carbon quaternary stereocenters.

Manganese(III) acetate mediated oxidative radical cyclizations have been used to synthesize a range of densely functionalized and sterically congested cyclopentane-lactones. A number of the resulting lactones contain vicinal all-carbon quaternary stereocenters adjacent to a tertiary benzylic stereocenter and are formed with high levels of stereocontrol.

New modes for the osmium-catalyzed oxidative cyclization.

The osmium-catalyzed oxidative cyclization of amino alcohol initiators formally derived from 1,4-dienes is an effective method for the construction of pyrrolidines, utilizing a novel reoxidant (4-nitropyridine N-oxide = NPNO). The cyclization of enantiopure syn- and anti-amino alcohols gives rise to enantiopure cis- and trans-2,5-disubstituted pyrrolidines, respectively. Moreover, the cyclization of bis-homoallylic amines bearing an exocyclic chelating group is shown to be a complementary method for trans-pyrrolidine formation.

Novel thienopyrrole glycogen phosphorylase inhibitors: synthesis, in vitro SAR and crystallographic studies.

Two series of novel thienopyrrole inhibitors of recombinant human liver glycogen phosphorylase a (GPa) which are effective in reducing glucose output from rat hepatocytes are described. Representative compounds have been shown to bind at the dimer interface site of the rabbit muscle enzyme by X-ray crystallography.