Bioconjugation

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(Redirected from
Conjugation (biochemistry)
)
For other uses, see Conjugation (disambiguation).

Bioconjugation is a chemical strategy to form a stable covalent link between two molecules, at least one of which is a biomolecule.

Overview

Function

Recent advances in the understanding of biomolecules enabled their application to numerous fields like medicine, diagnostics, biocatalysis and materials. Synthetically modified biomolecules can have diverse functionalities, such as tracking cellular events, revealing

quantum dots
.

Types of Conjugated Molecules

The most common types of bioconjugation include coupling of a small molecule (such as

Antibody-drug conjugates such as Brentuximab vedotin and Gemtuzumab ozogamicin are examples falling into this category.[5]

Protein-protein conjugations, such as the coupling of an antibody to an enzyme, or the linkage of protein complexes, is also facilitated via bioconjugations.[6][7]

Other less common molecules used in bioconjugation are

carbon nanotubes.[9]

Common Bioconjugation Reactions

amino acid residues, as well as modification of tryptophan residues and of the N- and C- terminus.[1][3][4]

However, these reactions often lack

Staudinger ligation with organic azides, copper-catalyzed Huisgen cycloaddition of azides, and strain promoted Huisgen cycloaddition of azides.[10][11][12][13]

On Natural Amino Acids

Reactions of lysines

The

isothiocyanates that undergo a similar mechanism (shown in the second and third reactions in Figure 1 below).[1] Benzoyl fluorides (shown in the last reaction below in Figure 1), which allows for lysine modification of proteins under mild conditions (low temperature, physiological pH), were recently proposed as an alternative to classically used lysine specific reagents.[14]

Figure 1. Bioconjugation strategies for lysine residues.png

Reactions of cysteines

Because free cysteine rarely occurs on protein surface, it is an excellent choice for chemoselective modification.

3-arylpropiolonitriles are used; the last reaction below in Figure 2).[17]

Figure 2. Bioconjugation strategies for cysteine residues.jpg

Reactions of tyrosines

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

aldehydes and anilines (the last reaction in Figure 3) was also described to be relatively tyrosine-selective under mild optimised reaction conditions.[19]

Figure 3. Bioconjugation strategies for tyrosine residues

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-terminal serine and threonine residues are able to generate N-terminal aldehyde, which can undergo further bioorthogonal reactions (shown in the first reaction in Figure 4). 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). Using pyridoxal phosphate (PLP), several N-terminal amino acids can undergo transamination to yield N-terminal aldehyde, such as glycine and aspartic acid (third reaction in Figure 4).

Figure 4. Bioconjugation strategies for N-terminus.jpg

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).

Figure 5. Bioconjugation strategies for C-terminus.jpg

Bioorthogonal Reactions: On Unique Functional Groups

Modification of ketones and aldehydes

A ketone or aldehyde can be attached to a protein through the oxidation of N-terminal serine residues or transamination with PLP. Additionally, they can be introduced by incorporating

acidic solution is often employed to accelerate the dehydration step.[2]

Figure 6. Bioconjugation strategies for targeting ketones and aldehydes.jpg

The introduction of nucleophilic catalyst can significantly enhance reaction rate (shown in Figure 7). For example, using

carbonyl becomes a highly populated protonated Schiff base.[20] 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.[20]
The key of this catalyst is that it can generate a reactive electrophile without competing with desired product.

Figure 7. Nucleophilic catalysis of oxime ligation.jpg

Recent developments that exploit proximal functional groups have enabled hydrazone condensations[21] to operate at 20 M−1s−1 at neutral pH while oxime condensations have been discovered which proceed at 500-10000 M−1s−1 at neutral pH without added catalysts.[22][23]

Staudinger ligation with azides

The

in vivo imaging, and other bioconjugation studies.[24][25][26][27]


Figure 8. Staudinger Ligation with Azides.jpg

Contrasting with the classic Staudinger reaction, Staudinger ligation is a

iminophosphorane intermediate, which will then give the amide-linkage under hydrolysis.[28]

Figure 9. Mechanism of Staudinger Ligation.jpg

Huisgen cyclization of azides

Main article:
Huisgen cycloaddition
Copper catalyzed Huisgen cyclization of azides

Azide has become a popular target for chemoselective protein modification, because they are small in size and have a favorable thermodynamic reaction potential. One such azide reactions is the [3+2] cycloaddition reaction with alkyne, but the reaction requires high temperature and often gives mixtures of regioisomers.

Figure 10. Copper-catalyzed cyclization of Azides.jpg

An improved reaction developed by chemist

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.[13]

Figure 11. Mechanism for Copper-catalyzed cyclization of Azides.jpg

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

Carolyn R. Bertozzi's lab developed a metal free [3+2] cycloaddition using strained cyclooctyne and azide. Cyclooctyne, which is the smallest stable cycloalkyne, can couple with azide through [3+2] cycloaddition, leading to two regioisomeric triazoles (Figure 12).[11] The reaction occurs readily 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 fluorine substituents on a cyclic alkyne can greatly accelerate the reaction rate.[2][29]

Figure 12. Strain promoted cycloaddition of azides and cyclooctynes.jpg

Transition Metal-Mediated Bioconjugation Reactions

copper-catalyzed [3 + 2] azide alkyne cycloaddition reaction, more and more diverse transition metal-mediated chemical transformations have been applied for bioconjugation reactions, introducing olefin metathesis, alkylation, C–H arylation, C–C, C–S, and C–N cross-coupling reactions.[30][31]

Alkylation

On Natural Amino Acids

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.

However, this method is limited to surface tryptophans and cysteines possibly because of steric constraints.[34]

  • Ir-catalyzed Lys and N-terminus (reductive) alkylation[35]

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). The reaction happens readily under physiologically relevant conditions and results in high conversion for various aromatic aldehydes.

  • Pd-catalyzed Tyr O-alkylation[36]

By using a pre-formed electrophilic π-allylpalladium(II) reagent derived from allylic acetate or carbamate precursors, selective allylic alkylation of tyrosines can be achieved in aqueous solution at room temperature and in the presence of cysteines.

  • Au-catalyzed Cys alkylation[37]

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. The reaction with peptides proceeds with high yields and is selective for cysteines over other nucleophilic residues.

However, the reactivity towards proteins is much decreased, potentially due to the coordination of gold to the protein backbone.

Arylation

On Natural Amino Acids

  • Trp arylation

Multiple methods have been reported to achieve tryptophan C–H arylation, where diverse electrophiles such as aryl halides[38][39] and aryl boronic acids[40] (an example shown below) have been used to transfer the aryl groups.

However, current tryptophan C–H arylation reaction conditions remain relatively harsh, requiring organic solvents, low pH and/or high temperatures.

  • Cys arylation

Free thiols has been considered unfavorable for Pd-mediated reactions due to Pd-catalyst decomposition.[41] However, PdII oxidative addition complexes (OACs) supported by dialkylbiaryl phosphine ligands have shown to work efficiently towards cysteine S-arylation.

The first example is the use of PdII OAC with

antibody-drug conjugates (ADCs). Changing the ligand to sSPhos supports the PdII complex to be sufficiently water soluble to achieve cysteine S-arylation under cosolvent-free aqueous conditions.[44]

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.[45]

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. The latter has been applied to selectively label surface lysine residues of a protein (forming PdII-protein OACs) and oligonucleotides (forming PdII-oligonucleotide OACs), which could then be linked to cysteine-containing peptides or proteins.[46]

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.[47]

Similar to cysteine, lysine N-arylation could be achieved through Pd OACs with different dialkylbiaryl phosphine ligands. 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. The reaction happens in mild conditions and is selective over most other nucleophilic amino acid residues.

On Unnatural Amino Acids

Pd-mediated

genetic code expansion
or post-translational modifications.

Examples of Applied Bioconjugation Techniques

Growth Factors

Bioconjugation of TGF-β to iron oxide nanoparticles and its activation through magnetic hyperthermia in-vitro has been reported.[58] This was done by using 1-(3-dimethylaminopropyl)ethylcarbodiimide combined with N-Hydroxysuccinimide to form primary amide bonds with the free primary amines on the growth factor. Carbon nanotubes have been successfully used in conjunction with bioconjugation to link TGF-β followed by an activation with near-infrared light.[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.[60]

See also

References

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