Bioconjugation is a chemical strategy for forming stable covalent bonds between two molecules, at least one of which is biomolecules.
Video Bioconjugation
Function
Recent advances in the understanding of biomolecules allow their applications to areas such as medicine and materials. Synthetically modified biomolecules can have multiple functions, such as tracking cellular events, revealing enzyme function, determining protein biodistribution, specific biomarker imaging, and sending drugs to targeted cells. Bioconjugation is an important strategy that links these modified biomolecules with different substrates.
Maps Bioconjugation
Synthesis
Bioconjugate synthesis involves a variety of challenges, ranging from the use of simple and non-specific fluorescent dye markers to the complex design of conjugate antibody drugs. As a result, various bioconjugation reactions - chemical reactions connecting two shared biomolecules - have been developed to modify proteins chemically. Common types of bioconjugation reactions are coupling of lysine amino acid residues, cysteine ​​residue couplings, tyrosine residue couplings, modification of tryptophan residues, and N- and C-terminus modifications.
However, these reactions often lack chemoselectivity and efficiency, as they depend on the presence of native amino acid residues, which are usually present in large amounts that inhibit selectivity. There is an increasing need for chemical strategies that can effectively attach to the site of synthetic molecules specific to proteins. One strategy is to first install a functional group unique to the protein, and then a bioorthogonal or click type reaction is used to pair the biomolecules with this unique functional group. Biooralogonal reactions that target non-native functional groups are widely used in chemical bioconjugation. Some important reactions are the modification of ketones and aldehydes, Staudinger ligation with azide, Huisgen cycloaddition Hiersgen which is enriched copper, and strains that promote Huisgen cycloaddition of azide.
General Bioconjugation Reactions
The most common bioconjugations are the coupling of small molecules (such as biotin or fluorescent dye) to proteins, or the conjugation of proteins, such as coupling antibodies to enzymes. Other less common molecules used in bioconjugation are oligosaccharides, nucleic acids, synthetic polymers such as polyethylene glycol, and carbon nanotubes. Conjugate antibodies 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 become important in nanotechnology applications such as bioconjugated quantum dots.
The reaction of lysine residue
Nucleophilic lysine residues are sites that are commonly targeted in protein bioconjugation, usually via an amine-reactive N-Hydroxysuccinimidyl (NHS) ester. To obtain an optimum deprotonated lysine residue amount, the pH of the aqueous solution should be below the pKa of the ammonium lysine group, which is about 10.5, so that the typical pH of the reaction is about 8 and 9. The general reagent for the coupling reaction is the NHS-ester (shown in the reaction first below in Figure 1 ), which reacts with the nucleophilic lysine via the lysine acylation mechanism. Other similar reagents are isocyanate and isothiocyanate which undergo the same mechanism (shown in the second and third reactions in Fig. 1 below). Benzoyl fluoride (shown in the last reaction below in Figure 1 ), which allows for the modification of lysine proteins under mild conditions (low temperature, physiological pH), was recently proposed as an alternative to specific lysine which is used specifically. reagent.
Reaksi residu sistein
Because free cysteine ​​rarely occurs on the surface of proteins, it is an excellent choice for kemoselective modifications. Under basic conditions, the cysteine ​​residue will be deprotonated to produce thiolate nuclei, which will react with soft electrophiles, such as maleimides and iodoacetamides (shown in the first two reactions in Fig. 2 below). As a result, carbon-sulfur bonds are formed. Another modification of cysteine ​​residue involves the formation of a disulfide bond (shown in the third reaction in Fig. 2 ). Reduced cysteine ​​residues react with exogenous disulfide, producing new disulfide bonds in proteins. Excess disulfide is often used to induce reactions, such as 2-thiopridone and 3-carboxy-4-nitrothiophenol. Electron-deficient alkynes are demonstrated to selectively react with protein cysteine ​​residues in the presence of other nucleophilic amino acid residues. Depending on the substitution of alkyne, these reactions can produce either split (when an alkynone derivative is used), or a hydrolytic stabilized bioconjugate (when 3-arylpropyolonitrile is used, the last reaction below at Fig. 2 ).
Reaksi residu tirosin
The tyrosine residue is relatively unreactive; therefore they have not become a popular target for bioconjugation. Recent developments have shown that tyrosine can be modified through electrophilic substitution reactions (EAS), and selectively for aromatic carbon adjacent to phenolic hydroxyl groups. This becomes very useful in case that cysteine ​​residues can not be targeted. In particular, diazonium is effectively paired with tyrosine residues (diazonium salts shown as reagents in the first reaction in Figure 3 below), and the electron-pulling substituents in 4-position diazonium salts can effectively increase the efficiency of the reaction. Cyclic diazodicarboxyamide derivatives such as 4-Phenyl-1,2,4-triazole-3,5-dione (PTAD) are reported for selective bioconjugation on tyrosine residues (second reaction in Figure 3 below). The three-mannich-type reaction with aldehydes and aniline (the last reaction in Fig. 3 ) is also described as a relatively tyrosine-selective relative under mild optimized reaction conditions.
Reactions from N- and C-termini
Since natural amino acid residues usually exist in large numbers, it is often difficult to modify a single site. Strategies that target protein termini have been developed, as they greatly improve the selectivity of protein modification sites. One of these N-termini modifications involves the functionalization of terminal amino acids. Oxidation of serine residues and N-terminal threonine are capable of producing an N-terminal aldehyde, which may undergo further bioortogonal reactions (shown in the first reaction in Fig. 4 ). Another type of modification involves the condensation of the N-terminal cysteine ​​with the aldehyde, yielding a stable thiazolidine at high pH (the second reaction in Fig. 4 ). Using pyridoxal phosphate (PLP), some N-terminal amino acids can undergo transamination to produce N-terminal aldehydes, such as glycine and aspartic acid (the third reaction in Figure 4 ).
An example of C-termini modification is the original chemical ligation (NCL), which is a coupling between the C-terminal thioester and the systeine ​​N-terminal ( Figure 5 ).
Bioortogonal reaction
Modify ketones and aldehydes
The ketone or aldehyde may be attached to the protein through the oxidation of N-terminal serine residues or transamination with PLP. In addition, they can be introduced by incorporating natural amino acids via the Tirrell method or the Schultz method. They will then selectively condense alkoxyamine and hydrazine, producing an oxime and a hydrazone derivative (shown in the first and second reactions, respectively, in Figure 6 ). This reaction is very chemoselective in terms of protein bioconjugation, but the reaction rate is slow. Mechanical studies have shown that the rate determinant step is the dehydration of tetrahedral intermediates, so a mild acid solution is often used to accelerate the dehydration step.
The introduction of a nucleophilic catalyst can significantly increase the reaction rate (shown in Figure 7 ). For example, using aniline as a nucleophilic catalyst, the less-populated protonated carbony becomes a very solid protonated Schiff base. In other words, it produces a high concentration of reactive electrophiles. The oxygen ligaments can then occur easily, and it has been reported that the rate increases up to 400 times under mild acid conditions. The key of this catalyst is that it can produce reactive electrophiles without competing with the desired product.
Recent developments that exploit proximal functional groups have allowed the condensation of hydrazon to operate at 20 M -1 s -1 at neutral pH while condensation oxime has been found that lasts at 500-10000. M -1 s -1 at neutral pH without additional catalyst.
Staudinger Ligation with Azide
Staudinger ligations of azide and phosphine have been widely used in the field of chemical biology. Because it is capable of forming stable amide bonds in living cells and animals, it has been applied to cell membrane modification, in vivo imaging, and other bioconjugation studies.
In contrast to the classical Staudinger's reaction, Staudinger ligation is a second-order reaction where the rate-limiting step is the formation of phosphazide (specific reaction mechanisms are shown in Figure 9 ). The first triphenylphosphine reacts with azide to produce azaylide through a four-membered transition state, and then an intramolecular reaction leads to iminophosphoric intermediates, which will then give the amide link under hydrolysis.
Huisgen Siklisasi dari Azides
Tembaga mengkatalisasi Huisgen Siklisasi dari Azides
Azide has become a popular target for the modification of kemoselective proteins, because they are small and have the potential for beneficial thermodynamic reactions. One such azide reaction is a cyclic reaction [3 2] with an alkaline, but the reaction requires a high temperature and often gives the mixture of the regioisomer.
The enhanced reaction developed by chemist Karl Barry Sharpless involves a copper (I) catalyst, which azide pairs with alkyne terminals which only provide 1.4 replaceable 1,2,3 triazoles in high yield (shown below in Fig. 11 ). Mechanical studies show a gradual reaction. The Cu (I) first pair with acetylenes, and then reacts with azide to produce a six-membered intermediate. This process is very strong which occurs at a pH ranging from 4 to 12, and copper (II) sulfate is often used as a catalyst in the presence of reducing agents.
Strain Promoted Huisgen Siklisasi dari Azides
Although Staudinger ligation is a suitable bioconjugation of living cells without major toxicity, the sensitivity of phosphine to air oxidation and its poor solubility in water significantly inhibits its efficiency. Copper (I) azide-alkyne clutch catalyst has a reasonable reaction rate and efficiency under physiological conditions, but copper raises significant toxicity and sometimes interferes with the function of proteins in living cells. In 2004, lab chemist Carolyn R. Bertozzi developed a free metal cyclic [3 2] using a strain of cyclooctyne and azide. Cyclooctyne, which is the smallest stable cycloalkyne, can pair with azide via cyclic [3 2], leading to two regioisomeric triazoles ( Figure 12 ). The reaction easily occurs at room temperature and therefore can be used to effectively modify living cells without negative effects. It has also been reported that the installation of a fluorine substituent in cyclic alkyl can greatly speed up the rate of reaction.
See also
- Immunofluorescence
- Biomolecular engineering
References
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