Felix F. Schumacher

Polymeric Dibromomaleimides As Extremely Efficient Disulfide Bridging Bioconjugation and Pegylation Agents

A series of dibromomaleimides have been shown to be very efficacious at insertion into peptidic disulfide bonds. This conjugation proceeds with a stoichiometric balance of reagents in buffered solutions in less than 15 min to give discrete products while maintaining the disulfide bridge and thus peptide conformation. The insertion is initiated by disulfide reduction using a water-soluble phosphine, tris(2-carboxyethyl)phosphine (TCEP) which allows for subsequent substitution of the two maleimide bromides by the generated thiols. Reaction of salmon calcitonin (sCT) with 2,3-dibromomaleimide (1.1 excess) in the presence of TCEP (1.1 equiv) in aqueous solution at pH 6.2 gives complete production of a single conjugate which requires no workup. A linear methoxy poly(ethylene glycol) (PEG) was functionalized via a Mitsunobu reaction and used for the successful site-specific and rapid pegylation of sCT. This reaction occurs in 15 min with a small stoichiometry excess of the pegylating agent to give insertion at the disulfide with HPLC showing a single product and MALDI-ToF confirming conjugation. Attempts to use the group in a functional ATRP polymerization initiator led to polymerization inhibition. Thus, in order to prepare a range of functional polymers an indirect route was chosen via both azide and aniline functional initiators which were converted to 2,3-dibromomaleimides via appropriate reactions. For example, the azide functional polymer was reacted via a Huisgen CuAAC click reaction to an alkyne functional 2,3-dibromomaleimide. This new reagent allowed for the synthesis of conjugates of sCT with comb polymers derived from PEG methacrylic monomers which in addition gave appropriate cloud points. This reaction represents a highly efficient polymer conjugation method which circumvents problems of purification which normally arise from having to use large excesses of the conjugate. In addition, the tertiary structure of the peptide is efficiently maintained.

In situ maleimide bridging of disulfides and a new approach to protein PEGylation.

The introduction of non-natural entities into proteins by chemical modification has numerous applications in fundamental biological science and for the development and manipulation of peptide and protein therapeutics. The reduction of native disulfide bonds provides a convenient method to access two nucleophilic cysteine residues that can serve as ideal attachment points for such chemical modification. The optimum bioconjugation strategy utilizing these cysteine residues should include the reconstruction of a bridge to mimic the role of the disulfide bond, maintaining structure and stability of the protein. Furthermore, the bridging chemical modification should be as rapid as possible to prevent problems associated with protein unfolding, aggregation, or disulfide scrambling. This study reports on an in situ disulfide reduction-bridging strategy that ensures rapid sequestration of the free cysteine residues in a bridge, using dithiomaleimides. This approach is then used to PEGylate the peptide hormone somatostatin and retention of biological activity is demonstrated.

Tunable reagents for multi-functional bioconjugation: reversible or permanent chemical modification of proteins and peptides by control of maleimide hydrolysis

Controlling maleimide hydrolysis allows the modular construction of bromomaleimide-mediated bioconjugates which are either stable or cleavable in an aqueous, thiol-mediated reducing environment.

Acid-cleavable thiomaleamic acid linker for homogeneous antibody–drug conjugation

Homogeneous antibody–drug conjugation is affected using a novel thiomaleamic acid linker that is stable at physiological temperature and pH, but quantitatively cleaves at lysosomal pH to release the drug payload.

Bromopyridazinedione-mediated protein and peptide bioconjugation

Bromopyridazinedione-mediated bioconjugation to a cysteine containing protein and a disulfide containing peptide is reported. These bioconjugates exhibit excellent hydrolytic stability and cleave in an aqueous, thiol-mediated reducing environment.

Zinc Binding to the Tyr402 and His402 Allotypes of Complement Factor H: Possible Implications for Age-Related Macular Degeneration

The Tyr402His polymorphism of complement factor H (FH) with 20 short complement regulator (SCR) domains is associated with age-related macular degeneration (AMD). How FH contributes to disease pathology is not clear. Both FH and high concentrations of zinc are found in drusen deposits, the key feature of AMD. Heterozygous FH is inhibited by zinc, which causes FH to aggregate. Here, zinc binding to homozygous FH was studied. By analytical ultracentrifugation, large amounts of oligomers were observed with both the native Tyr402 and the AMD-risk His402 homozygous allotypes of FH and both the recombinant SCR-6/8 allotypes with Tyr/His402. X-ray scattering also showed that both FH and SCR-6/8 allotypes strongly aggregated at > 10 μM zinc. The SCR-1/5 and SCR-16/20 fragments were less likely to bind zinc. These observations were supported by bioinformatics predictions. Starting from known zinc binding sites in crystal structures, we predicted 202 putative partial surface zinc binding sites in FH, most of which were in SCR-6. Metal site prediction web servers also suggested that SCR-6 and other domains bind zinc. Predicted SCR-6/8 dimer structures showed that zinc binding sites could be formed at the protein–protein interface that would lead to daisy-chained oligomers. It was concluded that zinc binds weakly to FH at multiple surface locations, most probably within the functionally important SCR-6/8 domains, and this explains why zinc inhibits FH activity. Given the high pathophysiological levels of bioavailable zinc present in subretinal deposits, we discuss how zinc binding to FH may contribute to deposit formation and inflammation associated with AMD.