One-pot thiol-amine bioconjugation to maleimides: simultaneous stabilisation and dual functionalisation.

Maleimide chemistry is widely used in the site-selective modification of proteins. However, hydrolysis of the resultant thiosuccinimides is required to provide robust stability to the bioconjugates. Herein, we present an alternative approach that affords simultaneous stabilisation and dual functionalisation in a one pot fashion.

Optimised approach to albumin-drug conjugates using monobromomaleimide-C-2 linkers.

Conjugation of therapeutics to human serum albumin (HSA) using bromomaleimides represents a promising platform for half-life extension. We show here that the Cys-34 crevice substantially reduces the rate of serum stabilising maleimide hydrolysis in these conjugates, necessitating reagent optimisation. This improved reagent design is applied to the construction of an HSA-paclitaxel conjugate, preventing drug loss during maleimide hydrolysis.

A new class of recombinant human albumin with multiple surface thiols exhibits stable conjugation and enhanced FcRn binding and blood circulation.

Human serum albumin is an endogenous ligand transport protein whose long circulatory half-life is facilitated by engagement with the human cellular recycling neonatal Fc receptor (hFcRn). The single free thiol located at Cys-34 in domain I of albumin has been exploited for monoconjugation of drugs. In this work, we increased the drug-to-albumin ratio potential by engineering recombinant human albumin (rHSA) variants with varying hFcRn affinity to contain three free, conjugation-competent cysteines.

Chemoselective Installation of Amine Bonds on Proteins through Aza-Michael Ligation.

Chemical modification of proteins is essential for a variety of important diagnostic and therapeutic applications. Many strategies developed to date lack chemo- and regioselectivity as well as result in non-native linkages that may suffer from instability in vivo and adversely affect the protein's structure and function. We describe here the reaction of N-nucleophiles with the amino acid dehydroalanine (Dha) in a protein context.

Albumin-based drug delivery using cysteine 34 chemical conjugates - important considerations and requirements.

The long blood circulation time of albumin has been clinically utilized as a half-life extension technology for improved drug performance. The availability of one free thiol for site-selective chemical conjugation offers an alternative approach to current genetic fusion and association-based products. This special report highlights important factors for successful conjugation that allows the reader to design and evaluate next-generation albumin conjugates.

Engineered Polymer-Transferrin Conjugates as Self-Assembling Targeted Drug Delivery Systems.

Polymer-protein conjugates can be engineered to self-assemble into discrete and well-defined drug delivery systems, which combine the advantages of receptor targeting and controlled drug release. We designed specific conjugates of the iron-binding and transport protein, transferrin (Tf), to combine the advantages of this serum-stable protein as a targeting agent for cancer cells with self-assembling polymers to act as carriers of cytotoxic drugs. Tf variants were expressed with cysteine residues at sites spanning different regions of the protein surface, and the polymer conjugates grown from these variants were compared with polymer conjugates grown from nonselectively derivatized sites on native Tf.

A platform for efficient, thiol-stable conjugation to albumin's native single accessible cysteine.

Herein we report the use of bromomaleimides for the construction of stable albumin conjugates via conjugation to its native, single accessible, cysteine followed by hydrolysis. Advantages over the classical maleimide approach are highlighted in terms of quantitative hydrolysis and absence of undesirable retro-Michael deconjugation.