Enzymatic Modification of N-Terminal Proline Residues Using Phenol Derivatives

Abstract

A convenient enzymatic strategyis reported for the modification of proline residues in the N-terminal positions of proteins. Using a tyrosinase enzyme isolated from Agaricus bisporus(abTYR), phenols and catechols are oxidized to highly reactive o-quinone intermediates that then couple to N-terminal proline residues in high yield. Key advantages of this bioconjugation method include (1) the use of air-stable precursors that can be prepared on large scale if needed, (2) mild reaction conditions, including low temperatures, (3) the targeting of native functional groups that can be introduced readily on most proteins, and (4) the use of molecular oxygen as the sole oxidant. This coupling strategy was successfully demonstrated for the attachment of a variety of phenol-derivatized cargo molecules to a series of protein substrates, including self-assembled viral capsids, enzymes, and a chitin binding domain (CBD). The ability of the CBD to bind to the surfaces of yeast cells was found to be unperturbed by this modification reaction.<br>

Site-Specific Bioconjugation through Enzyme-Catalyzed Tyrosine–Cysteine Bond Formation

“…This product contrasts with the previously observed structures resulting from proline and aniline additions to o -quinone species, where the addition occurs at the 6-position and the product tends to remain in the oxidized quinone form ( Figure 1 a). 12 − 14 MS data confirmed that the thiol addition products on larger biomolecules also existed predominantly in the reduced catechol form.…”

“… 21 Work from our lab has shown that abTYR and a new tyrosinase derived from Bacillus megatarium can activate phenols in both small-molecule and protein substrates for coupling to N-terminal proline residues and anilines ( Figure 1 a, top and bottom reaction pathways). 14 , 22 Taken together, these studies highlight the utility of tyrosinase enzymes to generate transient o -quinone electrophiles in dilute aqueous solutions, allowing highly chemoselective couplings that are not realized at the higher substrate concentrations evaluated previously.…”

“…A recent breakthrough in this chemistry involves the use of the tyrosinase enzymes to generate the same reactive o -quinone electrophiles using simple phenols and oxygen. 14 The small amount of molecular oxygen that is dissolved in the buffers is sufficient for the reaction to proceed, and water is the only byproduct. This reaction addresses two important limitations of the previous versions of this chemistry.…”

Reaction Landscape and Bioconjugation Profile of Tyrosinase Generated Quinones

“…mol -1 ) of exogenous amine resulted in any appreciable product formation, Fig S7. This is in contrast to secondary amines that are known to participate in dopaquinone modification, including N-terminal proline 22 and piperazine-type scaffolds. 16 The inability of primary amines to form conjugates with dopaquinones is puzzling.…”

Protein–Protein Conjugates: Tyrosine Delivers

“…This builds upon the team’s previous experience in utilizing quinone functionality, formed in situ, for reactions with other nucleophiles present on biomolecules such as proline residues and anilines. 3 , 4 While most proteins typically contribute a cysteine or lysine residue as the nucleophilic component of a bioconjugation reaction, the formation of an electrophilic ortho-quinone represents an interesting umpolung approach with the potential to expand the protein bioconjugation chemical space.…”