Skip to main content
SpringerLink
Log in

Chemical Synthesis of Activity-Based Diubiquitin Probes

  • Protocol
  • First Online:
Cancer Gene Networks

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1513))

Abstract

Activity-based diubiquitin probes are highly useful in probing the deubiquitinase (DUB) activity and ubiquitin chain linkage specificity. Here we describe a detailed protocol to synthesize a new class of diubiquitin DUB probes. In this method, two ubiquitin moieties are connected through a linker resembling the native linkage in size and containing a Michael acceptor for trapping the DUB active-site cysteine. Detailed procedures for generating the linker molecule are also described.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Nijman SM, Luna-Vargas MP, Velds A et al (2005) A genomic and functional inventory of deubiquitinating enzymes. Cell 123:773–786. doi:10.1016/j.cell.2005.11.007

    Article  CAS  PubMed  Google Scholar 

  2. Borodovsky A, Kessler BM, Casagrande R et al (2001) A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14. EMBO J 20:5187–5196. doi:10.1093/emboj/20.18.5187

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Borodovsky A, Ovaa H, Kolli N et al (2002) Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family. Chem Biol 9:1149–1159. doi:10.1016/S1074-5521(02)00248-X

    Article  CAS  PubMed  Google Scholar 

  4. de Jong A, Merkx R, Berlin I et al (2012) Ubiquitin-based probes prepared by total synthesis to profile the activity of deubiquitinating enzymes. ChemBioChem 13:2251–2258. doi:10.1002/cbic.201200497

    Article  PubMed  PubMed Central  Google Scholar 

  5. Ekkebus R, van Kasteren SI, Kulathu Y et al (2013) On terminal alkynes that can react with active-site cysteine nucleophiles in proteases. J Am Chem Soc 135:2867–2870. doi:10.1021/ja309802n

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Hemelaar J, Borodovsky A, Kessler BM et al (2004) Specific and covalent targeting of conjugating and deconjugating enzymes of ubiquitin-like proteins. Mol Cell Biol 24:84–95. doi:10.1128/MCB.24.1.84-95.2004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Love KR, Pandya RK, Spooner E, Ploegh HL (2009) Ubiquitin C-terminal electrophiles are activity-based probes for identification and mechanistic study of ubiquitin conjugating machinery. ACS Chem Biol 4:275–287. doi:10.1021/cb9000348

    Article  PubMed  PubMed Central  Google Scholar 

  8. McGouran JF, Kramer HB, Mackeen MM et al (2012) Fluorescence-based active site probes for profiling deubiquitinating enzymes. Org Biomol Chem 10:3379–3383. doi:10.1039/c2ob25258a

    Article  CAS  PubMed  Google Scholar 

  9. Ovaa H, Kessler BM, Rolen U et al (2004) Activity-based ubiquitin-specific protease (USP) profiling of virus-infected and malignant human cells. Proc Natl Acad Sci U S A 101:2253–2258. doi:10.1073/pnas.0308411100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Haj-Yahya N, Hemantha HP, Meledin R et al (2014) Dehydroalanine-based diubiquitin activity probes. Org Lett 16:540–543. doi:10.1021/ol403416w

    Article  CAS  PubMed  Google Scholar 

  11. Iphofer A, Kummer A, Nimtz M et al (2012) Profiling ubiquitin linkage specificities of deubiquitinating enzymes with branched ubiquitin isopeptide probes. ChemBioChem 13:1416–1420. doi:10.1002/cbic.201200261

    Article  PubMed  Google Scholar 

  12. Li G, Liang Q, Gong P et al (2014) Activity-based diubiquitin probes for elucidating the linkage specificity of deubiquitinating enzymes. Chem Commun 50:216–218. doi:10.1039/c3cc47382a

    Article  CAS  Google Scholar 

  13. McGouran JF, Gaertner SR, Altun M et al (2013) Deubiquitinating enzyme specificity for ubiquitin chain topology profiled by di-ubiquitin activity probes. Chem Biol 20:1447–1455. doi:10.1016/j.chembiol.2013.10.012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Mulder MP, El Oualid F, ter Beek J, Ovaa H (2014) A native chemical ligation handle that enables the synthesis of advanced activity-based probes: diubiquitin as a case study. ChemBioChem 15:946–949. doi:10.1002/cbic.201402012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Little TL, Webber SE (1994) A simple and practical synthesis of 2-aminoimidazoles. J Org Chem 59:7299–7305. doi:10.1021/jo00103a021

    Article  CAS  Google Scholar 

Download references

Acknowledgment

This work was supported, in part, by the US NIH grants GM097468 and NS085509 to Z. Zhuang.

Author information

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Delaware, 214A Drake Hall, Newark, DE, 19716, USA

    Guorui Li, Libo Yuan & Zhihao Zhuang

Authors
  1. Guorui Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Libo Yuan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhihao Zhuang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Zhihao Zhuang .

Editor information

Editors and Affiliations

  1. Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, District of Columbia, USA

    Usha Kasid

  2. Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, District of Columbia, USA

    Robert Clarke

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media New York

About this protocol

Cite this protocol

Li, G., Yuan, L., Zhuang, Z. (2017). Chemical Synthesis of Activity-Based Diubiquitin Probes. In: Kasid, U., Clarke, R. (eds) Cancer Gene Networks. Methods in Molecular Biology, vol 1513. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6539-7_16

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6539-7_16

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6537-3

  • Online ISBN: 978-1-4939-6539-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation