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 and materials. Synthetically modified biomolecules can have diverse functionalities, such as tracking cellular events, revealing enzyme function, determining protein biodistribution, imaging specific biomarkers, and delivering drugs to targeted cells.[1][2][3][4] Bioconjugation is a crucial strategy that links these modified biomolecules with different substrates. Besides applications in biomedical research, bioconjugation has recently also gained importance in nanotechnology such as bioconjugated quantum dots.

Types of Conjugated Molecules

The most common types of bioconjugation include coupling of a small molecule (such as biotin or a fluorescent dye) to a protein. 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, is also common in bioconjugations.[6]

Other less common molecules used in bioconjugation are oligosaccharides, nucleic acids, synthetic polymers such as polyethylene glycol,[7] and carbon nanotubes.[8]

Common Bioconjugation Reactions

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] Various bioconjugation reactions have been developed to chemically modify proteins. Common types of bioconjugation reactions on proteins are coupling of lysine, cysteine, and tyrosine 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 chemoselectivity and efficiency, because they depend on the presence of native amino acids, which are 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 onto a protein, and then a bioorthogonal reaction is used to couple a 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 organic azides, copper-catalyzed Huisgen cycloaddition of azides, and strain promoted Huisgen cycloaddition of azides.[9][10][11][12]

On Natural Amino Acids

Reactions of lysines

The nucleophilic lysine residue is commonly targeted site in protein bioconjugation, typically through amine-reactive N-hydroxysuccinimidyl (NHS) esters.[3] To obtain optimal number of deprotonated lysine residues, the pH of the aqueous solution must be below the pKa of the lysine ammonium group, which 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 (shown in the first reaction below in Figure 1), which reacts with nucleophilic lysine through a lysine acylation mechanism. Other similar reagents are isocyanates and 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.[13]

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.[14] 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). As a result, a carbon-sulfur bond is formed. Another modification of cysteine residues involves the formation of disulfide bond (shown in the third reaction in Figure 2). The reduced cysteine residues react with exogenous disulfides, generating new a disulfide bond on the protein. An excess of disulfides is often used to drive the reaction, such as 2-thiopyridone and 3-carboxy-4-nitrothiophenol.[1][3] Electron-deficient alkynes were demonstrated to selectively react with cysteine residues of proteins in the presence of other nucleophilic amino acid residues. Depending on the alkyne substitution, these reactions can produce either cleavable (when alkynone derivatives are used),[15] or hydrolytically stable bioconjugates (when 3-arylpropiolonitriles are used; the last reaction below in Figure 2).[16]

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 electrophilic aromatic substitutions (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 (diazonium salt shown as reagent in the first reaction in Figure 3 below), and an electron withdrawing substituent in the 4-position of diazonium salt can effectively increase the efficiency of the reaction. Cyclic diazodicarboxyamide derivative like 4-Phenyl-1,2,4-triazole-3,5-dione (PTAD) were reported for selective bioconjugation on tyrosine residues (the second reaction in Figure 3 below).[17] A three-component Mannich-type reaction with aldehydes and anilines (the last reaction in Figure 3) was also described to be relatively tyrosine-selective under mild optimised reaction conditions.[18]

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 unnatural amino acids via the Tirrell method or Schultz method.[9] They will then selectively condense with an alkoxyamine and a hydrazine, producing oxime and hydrazone derivatives (shown in the first and second reactions, respectively, in Figure 6). 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 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 aniline as a nucleophilic catalyst, a less populated protonated carbonyl becomes a highly populated protonated Schiff base.[19] 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.[19] 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[20] 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.[21][22]

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.[23][24][25][26]


Figure 8. Staudinger Ligation with Azides.jpg

Contrasting with the classic Staudinger reaction, Staudinger ligation is a second order reaction in which the rate-limiting step is the formation of phosphazide (specific reaction mechanism shown in Figure 9). 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.[27]

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 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). The mechanistic study suggests a stepwise reaction.[12] 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.[12]

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 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 2004, chemist 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).[10] 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][28]

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

Transition Metal-Mediated Bioconjugation Reactions

Transition metal-based bioconjugation had been challenging due to the nature of biological conditions – aqueous solution, room temperature, mild pH, and low substrate concentrations – which are generally challenging for organometallic reactions. However, recently, besides 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.[29][30]

Alkylation

On Natural Amino Acids

  • Rh-catalyzed Trp and Cys alkylation[31][32]

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.[33]

  • Ir-catalyzed Lys and N-terminus (reductive) alkylation[34]

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[35]

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[36]

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[37][38] and aryl boronic acids[39] (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.[40] 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 RuPhos:[41] The PdII complex resulting from the oxidative addition of aryl halides or trifluoromethanesulfonates and using RuPhos as the ligand could chemoselectively modify cysteines in various buffer with 5% organic co-solvent under neutral pH. This method has been shown to modify peptides and proteins, achieve peptide macrocyclization (by using bis-palladium reagent and peptides with two unprotected cysteines)[42] and synthesizing 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.[43]

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.[44]

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.[45]

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.[46]

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 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.[48] Those reactions require the protein or peptide substrate bearing unnatural functional groups such as alkyne,[49][50][51] aryl halides,[52][53][54][55] and aryl boronic acids,[56] which can be achieved through 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.[57] 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.[58] 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.[59]

See also

References

  1. ^ a b c d e f g Stephanopoulos N, Francis MB (November 2011). "Choosing an effective protein bioconjugation strategy". Nature Chemical Biology. 7 (12): 876–884. doi:10.1038/nchembio.720. PMID 22086289.
  2. ^ a b c Tilley SD, Joshi NS, Francis MB (2008). "Proteins: Chemistry and Chemical Reactivity". Wiley Encyclopedia of Chemical Biology. pp. 1–16. doi:10.1002/9780470048672.wecb493. ISBN 978-0470048672.
  3. ^ a b c d e Francis MB, Carrico IS (December 2010). "New frontiers in protein bioconjugation". Current Opinion in Chemical Biology. 14 (6): 771–773. doi:10.1016/j.cbpa.2010.11.006. PMID 21112236.
  4. ^ a b Kalia J, Raines RT (January 2010). "Advances in Bioconjugation". Current Organic Chemistry. 14 (2): 138–147. doi:10.2174/138527210790069839. PMC 2901115. PMID 20622973.
  5. ^ Gerber HP, Senter PD, Grewal IS (2009). "Antibody drug-conjugates targeting the tumor vasculature: Current and future developments". mAbs. 1 (3): 247–253. doi:10.4161/mabs.1.3.8515. PMC 2726597. PMID 20069754. Archived from the original on February 2, 2014.
  6. ^ Koniev O, Wagner A (August 2015). "Developments and recent advancements in the field of endogenous amino acid selective bond forming reactions for bioconjugation". Chemical Society Reviews. 44 (15): 5495–5551. doi:10.1039/C5CS00048C. PMID 26000775.
  7. ^ Thordarson P, Le Droumaguet B, Velonia K (November 2006). "Well-defined protein-polymer conjugates--synthesis and potential applications". Applied Microbiology and Biotechnology. 73 (2): 243–254. doi:10.1007/s00253-006-0574-4. PMID 17061132. S2CID 23657616.
  8. ^ Yang W, Thordarson P (2007). "Carbon nanotubes for biological and biomedical applications". Nanotechnology. 18 (41): 412001. Bibcode:2007Nanot..18O2001Y. doi:10.1088/0957-4484/18/41/412001. S2CID 137867074.
  9. ^ a b Carrico IS, Carlson BL, Bertozzi CR (June 2007). "Introducing genetically encoded aldehydes into proteins". Nature Chemical Biology. 3 (6): 321–322. doi:10.1038/nchembio878. PMID 17450134.
  10. ^ a b Agard NJ, Prescher JA, Bertozzi CR (November 2004). "A strain-promoted [3 + 2] azide-alkyne cycloaddition for covalent modification of biomolecules in living systems". Journal of the American Chemical Society. 126 (46): 15046–15047. doi:10.1021/ja044996f. PMID 15547999.
  11. ^ Kolb HC, Finn MG, Sharpless KB (June 2001). "Click Chemistry: Diverse Chemical Function from a Few Good Reactions". Angewandte Chemie. 40 (11): 2004–2021. doi:10.1002/1521-3773(20010601)40:11<2004::AID-ANIE2004>3.0.CO;2-5. PMID 11433435.
  12. ^ a b c Rostovtsev VV, Green LG, Fokin VV, Sharpless KB (July 2002). "A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective "ligation" of azides and terminal alkynes". Angewandte Chemie. 41 (14): 2596–2599. doi:10.1002/1521-3773(20020715)41:14<2596::AID-ANIE2596>3.0.CO;2-4. PMID 12203546.
  13. ^ Dovgan I, Ursuegui S, Erb S, Michel C, Kolodych S, Cianférani S, Wagner A (May 2017). "Acyl Fluorides: Fast, Efficient, and Versatile Lysine-Based Protein Conjugation via Plug-and-Play Strategy". Bioconjugate Chemistry. 28 (5): 1452–1457. doi:10.1021/acs.bioconjchem.7b00141. PMID 28443656.
  14. ^ Fodje MN, Al-Karadaghi S (May 2002). "Occurrence, conformational features and amino acid propensities for the pi-helix". Protein Engineering. 15 (5): 353–358. doi:10.1093/protein/15.5.353. PMID 12034854.
  15. ^ Shiu HY, Chan TC, Ho CM, Liu Y, Wong MK, Che CM (2009). "Electron-deficient alkynes as cleavable reagents for the modification of cysteine-containing peptides in aqueous medium". Chemistry: A European Journal. 15 (15): 3839–3850. doi:10.1002/chem.200800669. PMID 19229937.
  16. ^ Koniev O, Leriche G, Nothisen M, Remy JS, Strub JM, Schaeffer-Reiss C, et al. (February 2014). "Selective irreversible chemical tagging of cysteine with 3-arylpropiolonitriles". Bioconjugate Chemistry. 25 (2): 202–206. doi:10.1021/bc400469d. PMID 24410136.
  17. ^ Ban H, Nagano M, Gavrilyuk J, Hakamata W, Inokuma T, Barbas CF (April 2013). "Facile and stabile linkages through tyrosine: bioconjugation strategies with the tyrosine-click reaction". Bioconjugate Chemistry. 24 (4): 520–532. doi:10.1021/bc300665t. PMC 3658467. PMID 23534985.
  18. ^ Joshi NS, Whitaker LR, Francis MB (December 2004). "A three-component Mannich-type reaction for selective tyrosine bioconjugation". Journal of the American Chemical Society. 126 (49): 15942–15943. doi:10.1021/ja0439017. PMID 15584710.
  19. ^ a b Dirksen A, Hackeng TM, Dawson PE (November 2006). "Nucleophilic catalysis of oxime ligation". Angewandte Chemie. 45 (45): 7581–7584. doi:10.1002/anie.200602877. PMID 17051631.
  20. ^ Kool ET, Park DH, Crisalli P (November 2013). "Fast hydrazone reactants: electronic and acid/base effects strongly influence rate at biological pH". Journal of the American Chemical Society. 135 (47): 17663–17666. doi:10.1021/ja407407h. PMC 3874453. PMID 24224646.
  21. ^ Schmidt P, Zhou L, Tishinov K, Zimmermann K, Gillingham D (October 2014). "Dialdehydes lead to exceptionally fast bioconjugations at neutral pH by virtue of a cyclic intermediate". Angewandte Chemie. 53 (41): 10928–10931. doi:10.1002/anie.201406132. PMID 25164607.
  22. ^ Schmidt P, Stress C, Gillingham D (June 2015). "Boronic acids facilitate rapid oxime condensations at neutral pH". Chemical Science. 6 (6): 3329–3333. doi:10.1039/C5SC00921A. PMC 5656983. PMID 29142692.
  23. ^ Lemieux GA, De Graffenried CL, Bertozzi CR (April 2003). "A fluorogenic dye activated by the staudinger ligation". Journal of the American Chemical Society. 125 (16): 4708–4709. doi:10.1021/ja029013y. PMID 12696879.
  24. ^ Laughlin ST, Baskin JM, Amacher SL, Bertozzi CR (May 2008). "In vivo imaging of membrane-associated glycans in developing zebrafish". Science. 320 (5876): 664–667. Bibcode:2008Sci...320..664L. doi:10.1126/science.1155106. PMC 2701225. PMID 18451302.
  25. ^ Saxon E, Bertozzi CR (March 2000). "Cell surface engineering by a modified Staudinger reaction". Science. 287 (5460): 2007–2010. Bibcode:2000Sci...287.2007S. doi:10.1126/science.287.5460.2007. PMID 10720325. S2CID 19720277.
  26. ^ Prescher JA, Dube DH, Bertozzi CR (August 2004). "Chemical remodelling of cell surfaces in living animals". Nature. 430 (7002): 873–877. Bibcode:2004Natur.430..873P. doi:10.1038/nature02791. PMID 15318217. S2CID 4371934.
  27. ^ Lin FL, Hoyt HM, van Halbeek H, Bergman RG, Bertozzi CR (March 2005). "Mechanistic investigation of the staudinger ligation". Journal of the American Chemical Society. 127 (8): 2686–2695. doi:10.1021/ja044461m. PMID 15725026.
  28. ^ Chang PV, Prescher JA, Sletten EM, Baskin JM, Miller IA, Agard NJ, et al. (February 2010). "Copper-free click chemistry in living animals". Proceedings of the National Academy of Sciences of the United States of America. 107 (5): 1821–1826. doi:10.1073/pnas.0911116107. PMC 2836626. PMID 20080615.
  29. ^ Rodríguez J, Martínez-Calvo M (August 2020). "Transition-Metal-Mediated Modification of Biomolecules". Chemistry: A European Journal. 26 (44): 9792–9813. doi:10.1002/chem.202001287. PMID 32602145. S2CID 220272489.
  30. ^ Vinogradova EV (2017-11-01). "Organometallic chemical biology: an organometallic approach to bioconjugation". Pure and Applied Chemistry. 89 (11): 1619–1640. doi:10.1515/pac-2017-0207. ISSN 1365-3075. S2CID 103469980.
  31. ^ Antos JM, Francis MB (August 2004). "Selective tryptophan modification with rhodium carbenoids in aqueous solution". Journal of the American Chemical Society. 126 (33): 10256–10257. doi:10.1021/ja047272c. PMID 15315433.
  32. ^ Kundu R, Ball ZT (May 2013). "Rhodium-catalyzed cysteine modification with diazo reagents". Chemical Communications. 49 (39): 4166–4168. doi:10.1039/c2cc37323h. PMID 23175246.
  33. ^ Sauthoff G (1996-01-01). "Intermetallics J. Am. Chem. Soc. 1996, 118, 934". Journal of the American Chemical Society. 118 (43): 10678. doi:10.1021/ja965445v. ISSN 0002-7863.
  34. ^ McFarland JM, Francis MB (October 2005). "Reductive alkylation of proteins using iridium catalyzed transfer hydrogenation". Journal of the American Chemical Society. 127 (39): 13490–13491. doi:10.1021/ja054686c. PMID 16190700.
  35. ^ Tilley SD, Francis MB (February 2006). "Tyrosine-selective protein alkylation using pi-allylpalladium complexes". Journal of the American Chemical Society. 128 (4): 1080–1081. doi:10.1021/ja057106k. PMID 16433516.
  36. ^ Chan AO, Tsai JL, Lo VK, Li GL, Wong MK, Che CM (February 2013). "Gold-mediated selective cysteine modification of peptides using allenes". Chemical Communications. 49 (14): 1428–1430. doi:10.1039/c2cc38214h. PMID 23322001.
  37. ^ Ruiz-Rodríguez J, Albericio F, Lavilla R (January 2010). "Postsynthetic modification of peptides: chemoselective C-arylation of tryptophan residues". Chemistry: A European Journal. 16 (4): 1124–1127. doi:10.1002/chem.200902676. PMID 20013969.
  38. ^ Schischko A, Ren H, Kaplaneris N, Ackermann L (February 2017). "Bioorthogonal Diversification of Peptides through Selective Ruthenium(II)-Catalyzed C-H Activation". Angewandte Chemie. 56 (6): 1576–1580. doi:10.1002/anie.201609631. PMID 28074503.
  39. ^ Williams TJ, Reay AJ, Whitwood AC, Fairlamb IJ (March 2014). "A mild and selective Pd-mediated methodology for the synthesis of highly fluorescent 2-arylated tryptophans and tryptophan-containing peptides: a catalytic role for Pd(0) nanoparticles?". Chemical Communications. 50 (23): 3052–3054. doi:10.1039/C3CC48481E. PMID 24516861.
  40. ^ Bong DT, Ghadiri MR (August 2001). "Chemoselective Pd(0)-catalyzed peptide coupling in water". Organic Letters. 3 (16): 2509–2511. doi:10.1021/ol016169e. PMID 11483047.
  41. ^ Vinogradova EV, Zhang C, Spokoyny AM, Pentelute BL, Buchwald SL (October 2015). "Organometallic palladium reagents for cysteine bioconjugation". Nature. 526 (7575): 687–691. doi:10.1038/nature15739. PMC 4809359. PMID 26511579.
  42. ^ Rojas AJ, Zhang C, Vinogradova EV, Buchwald NH, Reilly J, Pentelute BL, Buchwald SL (June 2017). "Divergent unprotected peptide macrocyclisation by palladium-mediated cysteine arylation". Chemical Science. 8 (6): 4257–4263. doi:10.1039/C6SC05454D. PMC 5635729. PMID 29081961.
  43. ^ Rojas AJ, Pentelute BL, Buchwald SL (August 2017). "Water-Soluble Palladium Reagents for Cysteine S-Arylation under Ambient Aqueous Conditions". Organic Letters. 19 (16): 4263–4266. doi:10.1021/acs.orglett.7b01911. PMC 5818991. PMID 28777001.
  44. ^ Rojas AJ, Wolfe JM, Dhanjee HH, Buslov I, Truex NL, Liu RY, et al. (October 2022). "Palladium-peptide oxidative addition complexes for bioconjugation". Chemical Science. 13 (40): 11891–11895. doi:10.1039/D2SC04074C. PMC 9580489. PMID 36320916.
  45. ^ Jbara M, Rodriguez J, Dhanjee HH, Loas A, Buchwald SL, Pentelute BL (May 2021). "Oligonucleotide Bioconjugation with Bifunctional Palladium Reagents". Angewandte Chemie. 60 (21): 12109–12115. doi:10.1002/anie.202103180. PMC 8143041. PMID 33730425.
  46. ^ Dhanjee HH, Saebi A, Buslov I, Loftis AR, Buchwald SL, Pentelute BL (May 2020). "Protein-Protein Cross-Coupling via Palladium-Protein Oxidative Addition Complexes from Cysteine Residues". Journal of the American Chemical Society. 142 (20): 9124–9129. doi:10.1021/jacs.0c03143. PMC 7586714. PMID 32364380.
  47. ^ Lee HG, Lautrette G, Pentelute BL, Buchwald SL (March 2017). "Palladium-Mediated Arylation of Lysine in Unprotected Peptides". Angewandte Chemie. 56 (12): 3177–3181. doi:10.1002/anie.201611202. PMC 5741856. PMID 28206688.
  48. ^ Li J, Chen PR (August 2012). "Moving Pd-mediated protein cross coupling to living systems". ChemBioChem. 13 (12): 1728–1731. doi:10.1002/cbic.201200353. PMID 22764130. S2CID 19601358.
  49. ^ Li J, Lin S, Wang J, Jia S, Yang M, Hao Z, et al. (May 2013). "Ligand-free palladium-mediated site-specific protein labeling inside gram-negative bacterial pathogens". Journal of the American Chemical Society. 135 (19): 7330–7338. doi:10.1021/ja402424j. PMID 23641876.
  50. ^ Li N, Lim RK, Edwardraja S, Lin Q (October 2011). "Copper-free Sonogashira cross-coupling for functionalization of alkyne-encoded proteins in aqueous medium and in bacterial cells". Journal of the American Chemical Society. 133 (39): 15316–15319. doi:10.1021/ja2066913. PMC 3184007. PMID 21899368.
  51. ^ Li N, Ramil CP, Lim RK, Lin Q (February 2015). "A genetically encoded alkyne directs palladium-mediated protein labeling on live mammalian cell surface". ACS Chemical Biology. 10 (2): 379–384. doi:10.1021/cb500649q. PMC 4340352. PMID 25347611.
  52. ^ Chalker JM, Wood CS, Davis BG (November 2009). "A convenient catalyst for aqueous and protein Suzuki-Miyaura cross-coupling". Journal of the American Chemical Society. 131 (45): 16346–16347. doi:10.1021/ja907150m. PMID 19852502.
  53. ^ Spicer CD, Davis BG (February 2011). "Palladium-mediated site-selective Suzuki-Miyaura protein modification at genetically encoded aryl halides". Chemical Communications. 47 (6): 1698–1700. doi:10.1039/C0CC04970K. PMID 21206952.
  54. ^ Spicer CD, Triemer T, Davis BG (January 2012). "Palladium-mediated cell-surface labeling". Journal of the American Chemical Society. 134 (2): 800–803. doi:10.1021/ja209352s. PMID 22175226.
  55. ^ Dumas A, Spicer CD, Gao Z, Takehana T, Lin YA, Yasukohchi T, Davis BG (April 2013). "Self-liganded Suzuki-Miyaura coupling for site-selective protein PEGylation". Angewandte Chemie. 52 (14): 3916–3921. doi:10.1002/anie.201208626. PMID 23440916.
  56. ^ Brustad E, Bushey ML, Lee JW, Groff D, Liu W, Schultz PG (2008-10-13). "A genetically encoded boronate-containing amino acid". Angewandte Chemie. 47 (43): 8220–8223. doi:10.1002/anie.200803240. PMC 2873848. PMID 18816552.
  57. ^ Azie O, Greenberg ZF, Batich CD, Dobson JP (June 2019). "Carbodiimide Conjugation of Latent Transforming Growth Factor β1 to Superparamagnetic Iron Oxide Nanoparticles for Remote Activation". International Journal of Molecular Sciences. 20 (13): 3190. doi:10.3390/ijms20133190. PMC 6651417. PMID 31261853.
  58. ^ Lin L, Liu L, Zhao B, Xie R, Lin W, Li H, et al. (May 2015). "Carbon nanotube-assisted optical activation of TGF-β signalling by near-infrared light". Nature Nanotechnology. 10 (5): 465–471. Bibcode:2015NatNa..10..465L. doi:10.1038/nnano.2015.28. PMID 25775150.
  59. ^ Lalli E, Sarti G, Boi C (2018). "Effect of the spacer arm on non-specific binding in membrane affinity chromatography". MRS Communications. 8 (1): 65–70. doi:10.1557/mrc.2018.4. S2CID 103064199.