Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Jun 16;143(23):8661-8668.
doi: 10.1021/jacs.1c02206. Epub 2021 Jun 1.

An Organometallic Strategy for Cysteine Borylation

Mary A Waddington  1 , Xin Zheng  2 , Julia M Stauber  1 , Elamar Hakim Moully  1 , Hayden R Montgomery  1 , Liban M A Saleh  1 , Petr Král  2   3   4 , Alexander M Spokoyny  1   5
Affiliations

An Organometallic Strategy for Cysteine Borylation

Mary A Waddington et al. J Am Chem Soc. .

Abstract

Synthetic bioconjugation at cysteine (Cys) residues in peptides and proteins has emerged as a powerful tool in chemistry. Soft nucleophilicity of the sulfur in Cys renders an exquisite chemoselectivity with which various functional groups can be placed onto this residue under benign conditions. While a variety of reactions have been successful at producing Cys-based bioconjugates, the majority of these feature sulfur-carbon bonds. We report Cys-borylation, wherein a benchtop stable Pt(II)-based organometallic reagent can be used to transfer a boron-rich cluster onto a sulfur moiety in unprotected peptides forging a boron-sulfur bond. Cys-borylation proceeds at room temperature and tolerates a variety of functional groups present in complex polypeptides. Further, the bioconjugation strategy can be applied to a model protein modification of Cys-containing DARPin (designed ankyrin repeat protein). The resultant bioconjugates show no additional toxicity compared to their Cys alkyl-based congeners. Finally, we demonstrate how the developed Cys-borylation can enhance the proteolytic stability of the resultant peptide bioconjugates while maintaining the binding affinity to a protein target.

PubMed Disclaimer

Conflict of interest statement

Notes

The authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.
A: Summary of selective C-S bond forming reactions for bioconjugation of unprotected peptides and proteins. Metal-mediated strategies result in thiol arylation with the transferred group highlighted in blue. B: Summary of selective S-B bond forming reactions.
Figure 2.
Figure 2.
A: Representative LC trace collected after 1 (1.2 equiv) and H2N-VKGALGVCG-CONH2 (5 mM) were allowed to react for 1.5 h at 25 °C in the presence of Tris•HCl buffer (30 mM) in dimethylformamide (DMF). Internal standard was produced through alkylation of H2N-VKGALGVCG-CONH2 (see SI section I). B: Proposed reaction scheme between 1 and cysteine-containing peptide C: Peptide substrate scope with isolated yields (%) and conversion (%).
Figure 3.
Figure 3.
LC-MS traces of 7b incubated with 100 excess A: hydrochloric acid B: potassium carbonate and C: 7a after 24 hrs at r.t. followed by 48 and 72 hrs at 37 °C (See SI Section VII for full details). D: The % cell viability assessed after four-hour incubation of 7b and 7c with Chinese Hamster Ovarian (CHO) cells at 5 μM, 10 μM and 50 μM concentration of analyte (See SI Section IX for full details).
Figure 4.
Figure 4.
A: Representation of the inclusion complex formed between m-carborane and β-cyclodextrin., B: ITC binding plot for carboranylated glutathione and β-cyclodextrin to yield an association constant (Ka = 1.47 × 104 ± 500 M−1) and binding stoichiometry (N = 1). C: ITC binding plot for phenyl-glutathione and β-cyclodextrin. D: ITC binding plot for unmodified glutathione and β-cyclodextrin.
Figure 5.
Figure 5.
Molecular dynamics simulations of A: the secondary binding pocket identified as an important docking site B: 2b binding with Proteinase K, C: 2a binding with Proteinase K and D: 2c binding with Proteinase K at 120 ns of equilibration. Proteinase K is represented using QuickSurf representation in VMD.
See this image and copyright information in PMC

References

    1. Hermanson GT Bioconjugate Techniques, 3rd ed.; Academic Press: San Diego, CA, 2013.
    1. Recent representative reviews on general methods: Stephanopoulos N & Francis MB Choosing an Effective Protein Bioconjugation Strategy. Nature Chem. Bio 2011, 7, 876–884. - PubMed
    2. McKay CS & Finn MG Click Chemistry in Complex Mixtures: Biorthogonal Bioconjugation. Chem. Biol 2014, 21, 1075–1101. - PMC - PubMed
    3. Boutureira O & Bernardes GJL Advances in Chemical Protein Modification. Chem. Rev 2015, 115, 2174–2195. - PubMed
    4. Albaba B & Metzler-Nolte N Organometallic-Peptide Bioconjugates: Synthetic Strategies and Medicinal Applications. Chem. Rev 2016, 116, 11797–11839. - PubMed
    5. Hu Q-Y; Berti F; Adamo R Towards the Next Generation of Biomedicines by Site-Selective Conjugation. Chem. Soc. Rev 2016, 45, 1691–1719. - PubMed
    6. Vinogradova EV Organometallic Chemical Biology: An Organometallic Approach to Bioconjugation. Pure & Appl. Chem 2017, 89, 1619–1640.
    7. Jbara M; Maity SK; Brik A Palladium in the Chemical Synthesis and Modification of Proteins. Angew. Chem., Int. Ed 2017, 56, 10644–10655. - PubMed
    8. Ohata J; Martin SC; Ball ZT Metal-Mediated Functionalization of Natural Peptides and Proteins: Panning for Bioconjugation Gold. Angew. Chem., Int. Ed 2019, 58, 6176–6199. - PubMed
    9. Conibear AC; Watson EE; Payne RJ; Becker CFW Native Chemical Ligation in Protein Synthesis and Semi-Synthesis. Chem. Soc. Rev 2019, 47, 9046–9068. - PubMed
    10. Isenegger PG & Davis BG Concepts of catalysis in site-selective protein modifications. J. Am. Chem. Soc 2020, 141, 8005–8013. - PMC - PubMed
    11. Latocheski E; Dal Forno GM; Ferreira TM; Oliveira BL; Bernardes GJL; Domingos JB Mechanistic insights into transition metal-mediated bioorthogonal uncaging reactions Chem. Soc. Rev 2020, 49, 7710–7729. - PubMed
    12. Szijj PA; Kostadinova KA; Spears RJ; Chudasama V Tyrosine Bioconjugation - An Emergent Alternative. Org. Biomol. Chem 2020, 18, 9018–9028. - PMC - PubMed
    13. Patra M & Gasser G Organometallic Compounds: An Opportunity for Chemical Biology? ChemBioChem 2021, 13, 1232–1252. - PubMed
    1. Lavis LD Teaching Old Dyes New Tricks: Biological Probes Built from Fluoresceins and Rhodamines. Annu. Rev. Biochem 2017, 86, 825–43. - PubMed
    1. Li Y Commonly Used Tag Combinations for Tandem Affinity Purification. Biotechnol. Appl. Biochem 2010, 55, 73–83. - PubMed
    1. Liu H; Bolleddula J; Nichols A; Tang L; Zhao Z; Prakash C Metabolism of Bioconjugate Therapeutics: Why, When and How? Drug Metab. Rev 2020, 52, 66–124. - PubMed

Publication types

MeSH terms