Chemical Modification of Proteins at Cysteine: Opportunities in Chemistry and Biology

Abstract

Chemical modification of proteins is a rapidly expanding area in chemical biology. Selective installation of biochemical probes has led to a better understanding of natural protein modification and macromolecular function. In other cases such chemical alterations have changed the protein function entirely. Additionally, tethering therapeutic cargo to proteins has proven invaluable in campaigns against disease. For controlled, selective access to such modified proteins, a unique chemical handle is required. Cysteine, with its unique reactivity, has long been used for such modifications. Cysteine has enjoyed widespread use in selective protein modification, yet new applications and even new reactions continue to emerge. This Focus Review highlights the enduring utility of cysteine in protein modification with special focus on recent innovations in chemistry and biology associated with such modifications.

Versatile Synthesis of Stable, Functional Polypeptides via Reaction with Epoxides

“…We chose lysine as a competing nucleophile since it is the most abundant nucleophile found in proteins, it is more widely used in synthetic polypeptides compared to histidine or cysteine, and it is known to compete with thiol and imidazole groups in protein alkylations. 27,28 Similar to results obtained in other Met alkylations, 15 we found that the Met residues in the copolymer could be alkylated chemoselectively with glycidyl azide in acidic media in the presence of a fourfold excess of amine groups (eq. 2).…”

Selective chemical protein modification

“…As the most robustly nucleophilic of the 20 canonical amino acids, the thiol of cysteine offers a unique reactive handle within proteins, a property exploited extensively in nature 1 . Although pH may need to be controlled, selective reaction at cysteine over other nucleophilic residues such as lysine and histidine can be achieved 14 , while the low abundance (o2%) of cysteine in proteins often allows for facile modification at a single site 3 . In addition to functionalization of a protein of interest, this can also allow ready mutational repositioning and codon reassignment (through Cys-Ser/Gly mutation) 15 .…”

“…The UAA dehydroalanine (Dha) can be used as a Michael acceptor and has found extensive use in protein modification, reacting rapidly with sulfur nucleophiles to generate alkyl cysteine analogues, offering an electrophilic alternative to nucleophilic reaction of cysteine 3 . This is particularly useful in examples where use of electrophilic alkylation of Cys is difficult to control or where appropriate electrophiles cannot be generated, and leads to a greater level of selectivity.…”

“…Potential transformations, if they are to be relevant, are moulded by the need for biologically ambient conditions (that is, o37°C, pH 6-8, aqueous solvent) so as not to disrupt protein architecture and/or function. Ideally, this should proceed with near total conversion to generate homogenous constructs [2][3][4] . The applications of modified proteins are many; they are as varied as the in vivo tracking of protein-fluorophore conjugates 5 to the polyethylene glycol (PEG)ylation of therapeutic proteins to reduce immunogenicity 6 , from the production of materials with novel properties 7 to probing the mechanism of pathological enzymes 8 .…”

Functional Modification of Thioether Groups in Peptides, Polypeptides, and Proteins

“…3,4 Numerous strategies, including biological 8 and chemical synthesis, 1,2 have been developed to either react with or modify natural residues, or replace them entirely, using highly selective processes. Many of these methods have focused on reaction at or replacement of L-cysteine (Cys), 9 L-methionine (Met), 8,10,11 and N-terminal residues 12,13 since these are often present in low abundance and thus can provide unique sites for functional modification. Most chemical strategies have focused on reactions at highly nucleophilic Cys thiol groups, where a variety of different types of modification are possible.…”