Bioconjugation
[8][9] Synthesis of bioconjugates involves a variety of challenges, ranging from the simple and nonspecific use of a fluorescent dye marker to the complex design of antibody drug conjugates.
[1][3][4] However, these reactions often lack chemoselectivity and efficiency, because they depend on the presence of native amino acids, which are present in large quantities that hinder selectivity.
[10][11][12][13] The nucleophilic lysine residue is commonly targeted site in protein bioconjugation, typically through amine-reactive N-hydroxysuccinimidyl (NHS) esters.
[15] Under basic condition, the cysteine residues will be deprotonated to generate a thiolate nucleophile, which will react with soft electrophiles, such as maleimides and iodoacetamides (shown in the first two reactions in Figure 2 below).
Another type of modification involves the condensation of N-terminal cysteine with aldehyde, generating thiazolidine that is stable at high pH (second reaction in Figure 4).
An example of C-termini modification is the native chemical ligation (NCL), which is the coupling between a C-terminal thioester and a N-terminal cysteine (Figure 5).
The triphenylphosphine first reacts with the azide to yield an azaylide through a four-membered ring transition state, and then an intramolecular reaction leads to the iminophosphorane intermediate, which will then give the amide-linkage under hydrolysis.
An improved reaction developed by chemist Karl Barry Sharpless involves the copper (I) catalyst, which couples azide with terminal alkyne that only give 1,4 substituted 1,2,3 triazoles in high yields (shown below in Figure 11).
Even though Staudinger ligation is a suitable bioconjugation in living cells without major toxicity, the phosphine's sensitivity to air oxidation and its poor solubility in water significantly hinder its efficiency.
[30][31] Using in situ generated RhII-carbenoid by activation of vinyl-substituted diazo compounds with Rh2(OAc)4, tryptophans and cysteines were shown to be selectively alkylated in aqueous media.
Imines formed from the condensation of aldehydes with lysines or the N-terminus can be reduced efficient by an water-stable [Cp*Ir(bipy)(H2O)]SO4 complex in the presence of formate ions (serving as the hydride source).
Cysteine-containing peptides have been shown to undergo 1,2-addition to allenes in the presence of gold(I) and/or silver(I) salts, producing hydroxyl substituted vinyl thioethers.
However, current tryptophan C–H arylation reaction conditions remain relatively harsh, requiring organic solvents, low pH and/or high temperatures.
[41] However, PdII oxidative addition complexes (OACs) supported by dialkylbiaryl phosphine ligands have shown to work efficiently towards cysteine S-arylation.
Changing the ligand to sSPhos supports the PdII complex to be sufficiently water soluble to achieve cysteine S-arylation under cosolvent-free aqueous conditions.
There are other applications of this method where the PdII complexes were generated as PdII-peptide OACs by introducing 4-halophenylalanine into peptides during SPPS to achieve peptide-peptide or peptide-protein ligation.
Alternate to directly oxidative addition to the peptide, the Pd OACs could also be transferred to the protein through amine-selective acylation reaction via NHS ester.
Another example of protein-protein cross-coupling is achieved through converting cysteine residues into an electrophilic S-aryl–Pd–X OAC by utilizing an intramolecular oxidative addition strategy.
Due to weaker nucleophilicity and slower reductive elimination rate compared to cysteine, the selection of supporting ligands is shown to be critical.
The bulky BrettPhos and t-BuBrettPhos ligands in conjunction with mildly basic sodium phenoxide have been used as the strategy to functionalize lysines on peptide substrates.
Pd-mediated Sonogashira, Heck, and Suzuki-Miyaura cross-coupling reactions have been applied widely to modify peptides and proteins, where diverse Pd reagents have been developed for the application in aqueous solutions.
[59] Typically, these reactions have involved the use of a crosslinker, but some of these add molecular space between the compound of interest and base material and in turn causes higher degrees of non-specific binding and unwanted reactivity.
