Bioconjugation is the chemical strategy that couples two biomolecules together. Recent advances in the understanding of biomolecules enabled their application to numerous fields like medicine and materials. Synthetically modified biomolecules can have diverse functionalities, such as tracking localization of cellular events, elucidating enzyme function, determining protein biodistribution, imaging specific biomarkers, delivering drugs to targeted cells 1,4,19,21. Bioconjugation is a crucial strategy that links these modified biomolecules with different substrates.
Synthesis of bioconjugates involves a variety of challenges, ranging from the simple and nonspecific installation of a fluorescent dye to the complex design of antibody drug conjugates 1,19. As a result, various bioconjugation reactions – chemical reactions connecting two biomolecules together – have been developed to chemically modify proteins. Common types of bioconjugation reactions are coupling of lysine amino acid residues (typically through amine-reactive succinimidyl esters), coupling of cysteine residues (via a sulfhydryl-reactive maleimide), coupling of tyrosine residues (through electrophilic aromatic substitutions(EAS)), modification of tryptophan residues, and modification of the N- and C- terminus 1,19,21.
However, these reactions often lack chemoselectivity and efficiency, because they depend on the presence of native amino acid residues, which usually present in large quantities that hinder selectivity. There is an increasing need for chemical strategies that can effectively attach synthetic molecules site specifically to proteins. One strategy is to first install a unique functional group on protein, and then a bioorthogonal or click type reaction is utilized to couple biomolecule with this unique functional group 1. The bioorthogonal reactions targeting non-native functional groups are widely used in bioconjugation chemistry. Some important reactions are modification of ketone and aldehydes, Staudinger ligation with azides, copper-catalyzed huisgen cyclization of azide, strain promoted huisgen cyclization of azide 3,6,11,14,15.
“The most common bioconjugations are coupling of a small molecule (such as biotin or a fluorescent dye) to a protein, or protein-protein conjugations, such as the coupling of an antibody to an enzyme. Other less common molecules used in bioconjugation are oligosaccharides, nucleic acids, synthetic polymers such as polyethylene glycol (a.k.a. PEG a.k.a. polyethylene oxide), and carbon nanotubes. Antibody-drug conjugates such as Brentuximab vedotin and Gemtuzumab ozogamicin are also examples of bioconjugation, and are an active area of research in the pharmaceutical industry.Recently, bioconjugation has also gained importance in nanotechnology applications such as bioconjugated quantum dots.”
Common Bioconjugation reactions
Reactions of lysine residues
The nucleophilic lysine residue is commonly targeted site in protein bioconjugaiton 19. The reactions are usually run under basic condition. However, to obtain optimal number of deprotonated lysine residue, the pH of the aqueous solution must below the pKa of lysine ammonium group that is around 10.5. So the typical pH of the reaction is about 8 and 9. The common reagent for the coupling reaction is NHS-ester, which reacts with nucleophilic lysine through a lysine acylation mechanism. Other similar reagents are isocyanates and isothiocyanates that undergo a similar mechanism 1.
Reactions of cysteine residues
Because free cysteine rarely occurs on protein surface, it is an excellent choice for chemoselective modification. Under basic condition, the cysteine residues will be deprotonated to generate a thiolate nucleophile, which will react with soft electrohpiles, such as malemides and iodoacetamides. As a result, a carbon-sulfur bond is formed. Another modification of cysteine residues involves the formation of disulfide bond. The reduced cysteine residues react with exogenous disulfides, generating new disulfides bond on protein. An excess of disulfides is often used to drive the reaction, such as 2-thiopyridone and 3-carboxy-4-nitrothiophenol 1,19.
Reactions of Tyrosine residues
Tyrosine residues are relatively unreactive; therefore they have not been a popular targets for bioconjugation. Recent development has shown that the tyrosine can be modified through EAS reactions, and it is selective for the aromatic carbon adjacent to the phenolic hydroxyl group 1. This becomes particularly useful in the case that cysteine residues cannot be targeted. Specifically, diazonium effectively couples with tyrosine residues, and an electron withdrawing substituent in the 4-position of diazonium salt can effective increase the efficiency of the reaction.
Reactions of N- and C- termini
Since natural amino acid residues are usually present in large quantities, it is often difficult to modify one single site. Strategies targeting the termini of protein have been developed, because they greatly enhanced the site selectivity of protein modification. One of the N- termini modifications involves the functionalization of the terminal amino acid. The oxidation of N-terminial serine and threonine residues are able to generate N-terminal aldehyde, which can undergo further bioorthogonal reactions. Another type of modification involves the condensation of N-terminial cysteine with aldehyde, generating thiazolidine that is stable at high pH. Using pyridoxal phosphate (PLP), several N-terminal amino acids can undergo transamination to yield N-terminal aldehyde, such as glycine and aspartic acid. An example of C-termini modification is the native chemical ligation (NCL), which is the coupling between a C-terminal thoester and a N-terminal cysteine.
Modification of ketones and aldehydes
A ketone or aldehyde can be introduced to a protein through oxidation of N-terminal serine residues or transamination with PLP. In addition, they can be introduced by incorporating unnatural amino acid on protein, such as Tirrell method and Schultz method 3. They will then selectively condense with hydrazine and alkoxyamine, leading to hydrazone and oxime derivatives. This reaction is highly chemoselective in terms of protein bioconjugation, but the reaction rate is slow. The mechanistic studies show that the rate determining step is the dehydration of tetrahedral intermediate, so a mild acidic solution are often employed to accelerate the dehydration step 4. The introduction of nucleophilic catalyst can significantly enhance reaction rate. For example, using aniline as a nucleophilic catalyst, a less populated protonated carbonyl becomes a highly populated protonated Schiff base 12. In other words, it generates a high concentration of reactive electrophile. The oxime ligation can then occur readily, and it has been reported that the rate increased up to 400 times under mild acidic condition 12. The key of this catalyst is that it can generate a reactive electrophile without competing with desired product.
Staudinger Ligation with Azides
The Staudinger ligation of azides and phosphine has been used extensively in field of chemical biology. Because it is able to form a stable amide bond in living cells and animals, it has been applied to modification of cell membrane, in vivo imaging, and other bioconjugation studies 2,5,7,9. Contrasting with the classic Staudinger reaction, Staudinger Ligation is a 2nd order reaction that the rate-limiting step is the formation of phosphazide. The triphenylphosphine first react 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 13.
Copper catalyzed Huisgen Cyclization of Azides
Azide has become a popular target for chemoselective protein modification, because they are small in size and has a favorable thermodynamic reaction potential. One of the aizde reactions is the [3+2] cycloaddition reaction with alkyne, but the reaction requires high temperature and often gives mixtures of regioisomers. An improved reaction developed by 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. The mechanistic study suggests a stepwise reaction 15. The Cu (I) first couples with acetylenes, and then it reacts with azide to generate a six membered intermediate. The process is very robust that it occurs at pH ranging from 4 to 12, and copper (II) sulfate is often used as a catalyst in the presence of a reducing agent 15.
Strain Promoted Huisgen Cyclization of Azides
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. The copper (I) catalyzed azide-alkyne coupling has reasonable reaction rate and efficiency under physiological conditions, but copper poses significant toxicity and sometimes interferes with protein functions in living cells. In Bertozzi’s lab, a metal free [3+2] cycloaddition was developed using strained cyclooctyne and azide. Cyclooctyne, which is the smallest stable alkyne, can couples with azide through [3+2] cycloaddition, leading to two regioisomeric triazoles 6. The reaction occurs readily in room temperature and therefore can be used to effectively modify living cells without negative effects. It has also been reported that the installation of fluorine substituents on cyclic alkyne can greatly accelerate the reaction rate 4,11.
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